Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR THE CONSTRUCTION AND USE OF
FUSION LIBRARIES
This patent application claims priority to U.S. provisional patent
applications
S Serial No. 60/150,004, filed on August 20, 1999, and Serial No. 60/209,130,
filed June 2,
2000.
FIELD OF THE INVENTION
This invention pertains to genetic libraries encoding NAM enzyme fusion
proteins
and methods of use to identify a nucleic acid of interest.
BACKGROUND OF THE INVENTION
Improvements in DNA technology and bioinformatics have enabled the raw
genomic sequences of a few microorganisms to be made available to the
scientific
community, and the sequencing of genomes of higher eukaryotes and mammals are
nearly
completed. The rapid accumulation of DNA sequences from various organisms
presents
tremendous potential scientific and commercial opportunities. However, in many
cases,
the available raw sequences cannot be translated into knowledge of their
encoded
biological, pharmaceutical or industrial usefulness. Thus, there is a need in
the art for
technologies that will efficiently, systematically, and maximally realize the
function and
utility of DNA sequences from both natural and synthetic sources.
Several general approaches to realize the potential functions of a given DNA
sequence have been reported. One approach, which is also the primary approach
in gene
and target discovery, is to rely on bioinformatic tools. Bioinformatics
software is
available from a number of companies specializing in organization of sequence
data into
computer databases. A researcher is able to compare uncharacterized nucleic
acid
sequences with the sequences of known genes in the database, thereby allowing
theories
to be proposed regarding the function of the nucleic acid sequence of an
encoded gene
product. However, bioinformatics software can be expensive, often requires
extensive
training for meaningful use, and enables a researcher to only speculate as to
a possible
function of an encoded gene product. Moreover, an increasing number of DNA
sequences have been identified that show no sequence relationship to genes of
known
functions and new properties have been discovered for many so-called "known"
genes.
Therefore, bioinformatics provides a limited amount of information that must
be used
with caution. All informatics-predicted properties require experimental
approval.
Another approach for associating function with sequence data is to pursue
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experimental testing of orphan gene function. In previously described methods,
nucleic
acid sequences are expressed using any of a number of expression constructs to
obtain an
encoded peptide, which is then subjected to assays to identify a peptide
having a desired
property. An inherent difficulty with many of the previously described methods
is
correlating a target property with its coding nucleic acid sequence. In other
words, as
large collections of nucleic acid and peptide sequences are gathered and their
encoded
functions explored, it is increasingly difficult to identify and isolate a
coding sequence
responsible for a desired function.
The fundamental difficulties associated with working with large collections of
nucleic acid sequences, such as genetic libraries, are alleviated by linking
the expressed
peptide with the genetic material which encodes it. An approach of associating
a peptide
to its coding nucleic acid is the use of polysome display. Polysome display
methods
essentially comprise translating RNA in vitro and complexing the nascent
protein to its
corresponding RNA. The complex is constructed by manipulating the coding
sequence
1 S such that the ribosome does not release the nascent protein or the RNA. By
retrieving
proteins of interest, the researcher retrieves the corresponding RNA, and
thereby obtains
the coding DNA sequence after converting the RNA into DNA via known methods
such
as reverse transcriptase-coupled PCR. Yet, polysome display methods can be
carried out
only in vitro, are difficult to perform, and require an RNase-free
environment. Due to
alternative starting methionine codons and the less than perfect processive
nature of in
vitro translation machinery, this method is not applicable to large proteins.
In addition,
the RNA-protein-ribosome complex is unstable, thereby limiting screening
methods and
tools suitable for use with polysome display complexes.
Another commonly used method of linking proteins to coding nucleic acid
molecules for use with genetic libraries involves displaying proteins on the
outer surface
of cells, viruses, phages, and yeast. By expressing the variant protein as,
for example, a
component of a viral coat protein, the protein is naturally linked to its
coding DNA
located within the viral particle or cellular host, which can be easily
isolated. The DNA is
then purified and analyzed. Other systems for associating a protein with a DNA
molecule
in genetic library construction have been described in, for example,
International Patent
Applications WO 93/08278, WO 98/37186, and WO 99/11785. Yet, these approaches
have features that are not most desirable. First, the expressed protein and
the
corresponding cDNA are non-covalently bound. The resulting complex is not
stable or
suitable for many selection procedures. Second, the display systems by design
are
restricted to either in vitro or prokaryotic heterologous expression systems,
which may not
provide necessary protein modification or folding machinery for the study of
eukaryotic
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peptides. Incorrectly folded or modified proteins often lack the native
function of desired
proteins and are often very unstable. Third, if displayed on the surface of a
biological
particle, the expressed proteins often undergo unwanted biological selections
intrinsic to
the displayed systems. For example, in the case of display proteins on
bacterial viruses,
e.g., bacteriophage, the expressed protein will be assembled as part of
bacterial virus coat
proteins and displayed on the surface of the bacterial virus. Interactions of
the bacterial
virus-bound variant protein with the surrounding environment and incorporation
of the
protein into the bacterial viral coat can damage the conformation and activity
of the
variant protein. Moreover, even if the protein is incorporated into the
bacterial viral
capsid, the display protein may not be in a correct geometrical or
stoichiometrical form,
which is required for its activity. Fourth, construction of large surface-
display libraries
using biological particles is time intensive, and the researcher must take
precautions to
ensure that the biological particle, i.e., virus or phage, remains viable.
Fifth, it is known
that different hosts have different codon preferences when performing protein
translation.
For example, in prokaryotic systems, the expression systems used for bacterial
virus
display, there are at least five codons commonly recognized in mammalian cells
that are
not readily recognized by bacteria during protein translation. Thus, mammalian
sequences with these codons are not translated or are translated very
inefficiently in
bacteria, posing a significant negative selection.
In view of the above, there remains a need in the art for a genetic library
which
allows easy association of a variant or unknown peptide and its coding
sequence and
methods of use. The invention provides such a library and method. In addition,
the
present invention allows the identification of relevant proteins in the native
cellular
environment, which is a significant advantage of the use of eucaryotic
systems. These
and other advantages of the present invention, as well as additional inventive
features,
will be apparent from the description of the invention provided herein.
SUMMARY OF THE INVENTION
In accordance with the objects outlined herein, the present invention provides
libraries of fusion nucleic acids each comprising nucleic acid encoding a
nucleic acid
modification (NAM) enzyme, and nucleic acid encoding a candidate protein. At
least two
of the candidate proteins are different. In a preferred embodiment, the NAM
enzyme is a
Rep protein. Similarly, preferred embodiments utilize fusion nucleic acids
comprising
nucleic acids encoding presentation structures, nucleic acids encoding labels
or nucleic
acids encoding targeting sequences.
In an additional embodiment, the invention provides libraries of fusion
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polypeptides each comprising a NAM enzyme and a candidate protein, wherein at
least
two of the candidate proteins are different. In a preferred embodiment, the
NAM enzyme
is a Rep protein. Similarly, preferred embodiments utilize fusion polypeptides
comprising
presentation structures, labels or targeting sequences.
In a further embodiment, the invention provides libraries of expression
vectors
each comprising a fusion nucleic acid comprising a nucleic acid encoding a NAM
enzyme, a nucleic acid encoding a candidate protein, and an enzyme attachment
sequence
(EAS) that is recognized by the NAM enzyme. At least two of the candidate
proteins are
different. In a preferred embodiment, the NAM enzyme is a Rep protein.
Similarly,
preferred embodiments utilize fusion nucleic acids comprising nucleic acids
encoding
presentation structures, nucleic acids encoding labels or nucleic acids
encoding targeting
sequences. A preferred embodiment also utilizes EASs comprising at least 20
nucleotides.
In an additional embodiment, the invention provides libraries of nucleic
acid/protein (NAP) conjugates each comprising a fusion polypeptide comprising
a NAM
enzyme and a candidate protein. The NAP conjugates also comprise an expression
vector
comprising a fusion nucleic acid comprising a fusion nucleic acid comprising a
nucleic
acid encoding a NAM enzyme, a nucleic acid encoding a candidate protein, and
an
enzyme attachment sequence (EAS) that is recognized by the NAM enzyme. The EAS
and the NAM enzyme are covalently attached. At least two of the candidate
proteins are
different. In a preferred embodiment, the NAM enzyme is a Rep protein.
Similarly,
preferred embodiments utilize fusion nucleic acids comprising nucleic acids
encoding
presentation structures, nucleic acids encoding labels or nucleic acids
encoding targeting
sequences. A preferred embodiment also utilizes EASs comprising at least 20
nucleotides.
In a further aspect, the invention provides host cells comprising the
compositions
of the invention.
In an additional aspect, the invention provides libraries of eucaryotic host
cells
each comprising an expression vector comprising a fusion nucleic acid
comprising a
nucleic acid encoding a NAM enzyme, a nucleic acid encoding a candidate
protein, and
an enzyme attachment sequence (EAS) that is recognized by the NAM enzyme. At
least
two of the candidate proteins are different. In a preferred embodiment, the
NAM enzyme
is a Rep protein. Similarly, preferred embodiments utilize fusion nucleic
acids
comprising nucleic acids encoding presentation structures, nucleic acids
encoding labels
or nucleic acids encoding targeting sequences. A preferred embodiment also
utilizes
EASs comprising at least 20 nucleotides.
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In a further aspect, the invention provides libraries of eucaryotic host cells
each
comprising a nucleic acid/protein (NAP) conjugates. Each NAP comprises a
fusion
polypeptide comprising a NAM enzyme and a candidate protein. The NAP
conjugates
also comprise an expression vector comprising a fusion nucleic acid comprising
a nucleic
5 acid encoding a NAM enzyme, a nucleic acid encoding a candidate protein, and
an
enzyme attachment sequence (EAS) that is recognized by the NAM enzyme. The EAS
and the NAM enzyme are covalently attached. At least two of the candidate
proteins are
different. In a preferred embodiment, the NAM enzyme is a Rep protein.
Similarly,
preferred embodiments utilize fusion nucleic acids comprising nucleic acids
encoding
presentation structures, nucleic acids encoding labels or nucleic acids
encoding targeting
sequences. A preferred embodiment also utilizes EASs comprising at least 20
nucleotides.
In an additional aspect, the invention provides methods of screening
comprising
adding a library of NAP conjugates to at least one target molecule, and
determining the
binding of a NAP conjugate to the target.
In a further aspect, the invention provides methods of screening comprising
providing a library of host eucaryotic cells each comprising at least one NAP
conjugate
and screening the cells for an altered phenotype.
In an additional aspect, the invention provides methods of screening
comprising
providing a library of eucaryotic host cells each comprising at least one
expression vector,
and screening the host cells for an altered phenotype.
In further aspect, the invention provides methods of screening comprising
providing a library of eucaryotic host cells each comprising at least one
expression vector,
under conditions whereby a fusion polypeptide is produced and wherein at least
two of
the candidate proteins are different. The method further comprises lysing the
cells,
wherein the said EAS and the NAM enzyme are covalently attached to form a NAP
conjugate. A target molecule is added and the binding of the target to a NAP
conjugate is
determined.
DETAILED DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the nucleotide sequence of Rep78 isolated from adeno-
associated
virus 2.
Figure 2 depicts the amino acid sequence of Rep78 isolated from adeno-
associated
virus 2.
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Figure 3 depicts the nucleotide sequence of major coat protein A isolated from
adeno-associated virus 2.
Figure 4 depicts the amino acid sequence of major coat protein A isolated from
adeno-associated virus 2.
Figure 5 depicts the nucleotide sequence of a Rep protein isolated from adeno-
associated virus 4.
Figure 6 depicts the amino acid sequence of a Rep protein isolated from adeno-
associated virus 4.
Figure 7 depicts the nucleotide sequence of Rep78 isolated from adeno-
associated
virus 3B.
Figure 8 depicts the amino acid sequence of Rep78 isolated from adeno-
associated
virus 3B.
Figure 9 depicts the nucleotide sequence of a nonstructural protein isolated
from
adeno-associated virus 3.
Figure 10 depicts the amino acid sequence of a nonstructural protein isolated
from
adeno-associated virus 3.
Figure 11 depicts the nucleotide sequence of a nonstructural protein isolated
from
adeno-associated virus 1.
Figure 12 depicts the amino acid sequence of a nonstructural protein isolated
from
adeno-associated virus 1.
Figure 13 depicts the nucleotide sequence of Rep78 isolated from adeno-
associated virus 6.
Figure 14 depicts the amino acid sequence of Rep78 isolated from adeno-
associated virus 6.
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Figure 15 depicts the nucleotide sequence of Rep68 isolated from adeno-
associated virus 2.
Figure 16 depicts the amino acid sequence of Rep68 isolated from adeno-
associated virus 2.
Figure 17 depicts the nucleotide sequence of major coat protein A' (alt.)
isolated
from adeno-associated virus 2.
Figure 18 depicts the amino acid sequence of major coat protein A' (alt.)
isolated
from adeno-associated virus 2.
Figure 19 depicts the nucleotide sequence of major coat protein A" (alt.)
isolated
from adeno-associated virus 2.
Figure 20 depicts the amino acid sequence of major coat protein A" (alt.)
isolated
from adeno-associated virus 2.
Figure 21 depicts the nucleotide sequence of a Rep protein isolated from adeno-
associated virus 5.
Figure 22 depicts the amino acid sequence of a Rep protein isolated from adeno-
associated virus 5.
Figure 23 depicts the nucleotide sequence of major coat protein Aa (alt.)
isolated
from adeno-associated virus 2.
Figure 24 depicts the amino acid sequence of major coat protein Aa (alt.)
isolated
from adeno-associated virus 2.
Figure 25 depicts the nucleotide sequence of a Rep protein isolated from
Barbarie
duck parvovirus.
Figure 26 depicts the amino acid sequence of a Rep protein isolated from
Barbarie
duck parvovirus.
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Figure 27 depicts the nucleotide sequence of a Rep protein isolated from goose
parvovirus.
Figure 28 depicts the amino acid sequence of a Rep protein isolated from goose
parvovirus.
Figure 29 depicts the nucleotide sequence of NS 1 isolated from muscovy duck
parvovirus.
Figure 30 depicts the amino acid sequence of NS 1 isolated from muscovy duck
parvovirus.
Figure 31 depicts the nucleotide sequence of NS 1 isolated from goose
parvovirus.
Figure 32 depicts the amino acid sequence of NS 1 isolated from goose
parvovirus.
Figure 33 depicts the nucleotide sequence of non-structural protein 1 isolated
from
chipmunk parvovirus.
Figure 34 depicts the amino acid sequence of non-structural protein 1 isolated
from chipmunk parvovirus.
Figure 35 depicts the nucleotide sequence of non-structural protein isolated
from
the pig-tailed macaque parvovirus.
Figure 36 depicts the amino acid sequence of non-structural protein isolated
from
the pig-tailed macaque parvovirus.
Figure 37 depicts the nucleotide sequence of NS 1 isolated from a simian
parvovirus.
Figure 38 depicts the amino acid sequence of NS 1 protein isolated from a
simian
parvovtrus.
Figure 39 depicts the nucleotide sequence of a NS protein isolated from the
Rhesus
macaque parvovirus.
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Figure 40 depicts the amino acid sequence of a NS protein isolated from the
Rhesus macaque parvovirus.
S Figure 41 depicts the nucleotide sequence of a non-structural protein
isolated from
the B 19 virus.
Figure 42 depicts the amino acid sequence of a non-structural protein isolated
from
the B 19 virus.
Figure 43 depicts the nucleotide sequence of orf 1 isolated from the
Erythrovirus
B19.
Figure 44 depicts the amino acid sequence of the product of orf 1 isolated
from the
Erythrovirus B 19.
Figure 45 depicts the nucleotide sequence of U94 isolated from the human
herpesvirus 6B.
Figure 46 depicts the amino acid sequence of U94 isolated from the human
herpesvirus 6B.
Figure 47 depicts an enzyme attachment site for a Rep protein.
Figure 48 depicts the Rep 68 and Rep 78 enzyme attachment site found in
chromosome 19.
Figures 49A-49N depict preferred embodiments of the expression vectors of the
invention.
DETAILED DESCRIPTION
Significant effort is being channeled into screening techniques that can
identify
proteins relevant in signaling pathways and disease states, and to compounds
that can
effect these pathways and disease states. Many of these techniques rely on the
screening
of large libraries, comprising either synthetic or naturally occurring
proteins or peptides,
in assays such as binding or functional assays. One of the problems facing
high
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throughput screening technologies today is the difficulty of elucidating the
identification
of the "hit", i.e. a molecule causing the desired effect, against a background
of many
candidates that do not exhibit the desired properties.
The present invention is directed to a novel method that can allow the rapid
and
5 facile identification of these "hits". The present invention relies on the
use of nucleic acid
modification enzymes that covalently and specifically bind to the nucleic acid
molecules
comprising the sequence that encodes them. Proteins of interest (for example,
candidates
to be screened either for binding to disease-related proteins or for a
phenotypic effect) are
fused (either directly or indirectly, as outlined below) to a nucleic acid
modification
10 (NAM) enzyme. The NAM enzyme will covalently attach itself to a
corresponding NAM
attachment sequence (termed an enzyme attachment sequence (EAS)). Thus, by
using
vectors that comprise coding regions for the NAM enzyme and candidate proteins
and the
NAM enzyme attachment sequence, the candidate protein is covalently linked to
the
nucleic acid that encodes it upon translation. Thus, after screening,
candidates that
exhibit the desired properties can be quickly isolated using a variety of
methods such as
PCR amplification. This facilitates the quick identification of useful
candidate proteins,
and allows rapid screening and validation to occur.
Accordingly, the present invention provides libraries of nucleic acid
molecules
comprising nucleic acid sequences encoding fusion nucleic acids encoding a
nucleic acid
modification enzyme and a candidate protein. By "nucleic acid" or
"oligonucleotide" or
grammatical equivalents herein means at least two nucleosides covalently
linked
together. A nucleic acid of the present invention will generally contain
phosphodiester
bonds, although in some cases nucleic acid analogs are included that may have
alternate
backbones, particularly when the target molecule is a nucleic acid,
comprising, for
example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and
references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al.,
Eur. J.
Biochem. 81:579 ( 1977); Letsinger et al., Nucl. Acids Res. 14:3487 ( 1986);
Sawai et al,
Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988);
and
Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al.,
Nucleic
Acids Res. 19:1437 (1991); and U.S. Patent No. 5,644,048), phosphorodithioate
(Briu et
al., J. Am. Chem. Soc. 111:2321 ( 1989), O-methylphophoroamidite linkages (see
Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford
University
Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am.
Chem. Soc.
114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen,
Nature,
365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are
incorporated by
reference). Other analog nucleic acids include those with positive backbones
(Denpcy et
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11
al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.5.
Patent Nos.
5,386,023. 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al.,
Angew.
Chem. Intl. Ed. English 30:423 ( 1991 ); Letsinger et al., J. Am. Chem. Soc.
110:4470
(1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2
and 3, ASC
Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed.
Y.S.
Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett.
4:395
( 1994); Jeffs et al., J. Biomolecular NMR 34:17 ( 1994); Tetrahedron Lett. 3
7:743 ( 1996))
and non-ribose backbones, including those described in U.S. Patent Nos.
5,235,033 and
5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate
Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook.
Nucleic acids
containing one or more carbocyclic sugars are also included within the
definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. ( 1995) pp 169-176).
Several nucleic
acid analogs are described in Rawls, C & E News June 2, 1997 page 35. All of
these
references are hereby expressly incorporated by reference. These modifications
of the
ribose-phosphate backbone may be done to facilitate the addition of other
elements, such
as labels, or to increase the stability and half life of such molecules in
physiological
environments.
As will be appreciated by those in the art, all of these nucleic acid analogs
may
find use in the present invention. In addition, mixtures of naturally
occurring nucleic
acids and analogs can be made, or, alternatively, mixtures of different
nucleic acid
analogs, and mixtures of naturally occurring nucleic acids and analogs may be
made.
The nucleic acids may be single stranded or double stranded, as specified, or
contain portions of both double stranded or single stranded sequence. The
nucleic acid
may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid
contains
any combination of deoxyribo- and ribo-nucleotides, and any combination of
bases,
including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine
hypoxathanine,
isocytosine, isoguanine, etc. As used herein, the term "nucleoside" includes
nucleotides
and nucleoside and nucleotide analogs, and modified nucleosides such as amino
modified
nucleosides. In addition, "nucleoside" includes non-naturally occurring analog
structures.
Thus for example the individual units of a peptide nucleic acid, each
containing a base,
are referred to herein as a nucleoside.
The present invention provides libraries of nucleic acid molecules comprising
nucleic acid sequences encoding fusion nucleic acids. By "fusion nucleic acid"
herein is
meant a plurality of nucleic acid components (e.g., peptide coding sequences)
that are
joined together. The fusion nucleic acids preferably encode fusion
polypeptides, although
this is not required. By "fusion polypeptide" or "fusion peptide" or
grammatical
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equivalents herein is meant a protein composed of a plurality of protein
components, that
while typically unjoined in their native state, are joined by their respective
amino and/or
carboxyl termini through a peptide linkage to form a single continuous
polypeptide.
Plurality in this context means at least two, and preferred embodiments
generally utilize
S two components. It will be appreciated that the protein components can be
joined directly
or joined through a peptide linker/spacer as outlined below. In addition, it
should be
noted that in some embodiments, as is more fully outlined below, the fusion
nucleic acids
can encode protein components that are not fused; for example, the fusion
nucleic acid
may comprise an intron that is removed, leaving two non-associated protein
components,
although generally the nucleic acids encoding each component are fused.
Furthermore, as
outlined below, additional components such as fusion partners including
targeting
sequences, etc., can be used.
The fusion nucleic acids encode nucleic acid modification (NAM) enzymes and
candidate proteins. By "nucleic acid modification enzyme" or "NAM enzyme"
herein is
meant an enzyme that utilizes nucleic acids, particularly DNA, as a substrate
and
covalently attaches itself to nucleic acid enzyme attachment (EA) sequences.
The
covalent attachment can be to the base, to the ribose moiety or to the
phosphate moieties.
NAM enzymes include, but are not limited to, helicases, topoisomerases,
polymerases,
gyrases, recombinases, transposases, restriction enzymes and nucleases. As
outlined
below, NAM enzymes include natural and non-natural variants. Although many DNA
binding peptides are known, such as those involved in nucleic acid compaction,
transcription regulators, and the like, enzymes that covalently attach to
nucleic acids, i.e.,
DNA, in particular peptides involved with replication, are preferred. Some NAM
enzymes can form covalent linkages with DNA without nicking the DNA. For
example,
it is believed that enzymes involved in DNA repair recognize and covalently
attach to
nucleic acid regions, which can be either double-stranded or single-stranded.
Such NAM
enzymes are suitable for use in the fusion enzyme library. However, DNA NAM
enzymes that nick DNA to form a covalent linkage, e.g., viral replication
peptides, are
most preferred.
Preferably, the NAM enzyme is a protein that recognizes specific sequences or
conformations of a nucleic acid substrate and performs its enzymatic activity
such that a
covalent complex is formed with the nucleic acid substrate. Preferably, the
enzyme acts
upon nucleic acids, particularly DNA, in various configurations including, but
not limited
to, single-strand DNA, double-strand DNA, Z-form DNA, and the like.
Suitable NAM enzymes, include, but are not limited to, enzymes involved in
replication such as Rep68 and Rep78 of adeno-associated viruses (AAV), NS1 and
H-1 of
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parvovirus, bacteriophage phi-29 terminal proteins, the 55 Kd adenovirus
proteins, and
derivatives thereof.
In a preferred embodiment, the NAM enzyme is a Rep protein. Rep proteins
include, but are not limited to, Rep78, Rep68, and functional homologs thereof
found in
related viruses. Rep proteins, including their functional homologs, may be
isolated from a
variety of sources including parvoviruses, erythroviruse, herpesviruses, and
other related
viruses. One with ordinary skill in the art will appreciate that the natural
Rep protein can
be mutated or engineered with techniques known in the art in order to improve
its activity
or reduce its potential toxicity. Such experimental improvements may done in
conjunction with native or variants of their corresponding EAS. One of
preferred Rep
proteins is the AAV Rep protein. Adeno-associated viral (AAV) Rep proteins are
encoded by the left open reading frame of the viral genome. AAV Rep proteins,
such as
Rep68 and Rep78, regulate AAV transcription, activate AAV replication, and
have been
shown to inhibit transcription of heterologous promoters (Chiorini et al., J.
Virol., 68(2),
797-804 ( 1994), hereby incorporated by reference in its entirety). The Rep68
and Rep78
proteins act, in part, by covalently attaching to the AAV inverted terminal
repeat (Prasad
et al., Virology, 229, 183-192 (1997); Prasad et al., Virology, 214:360
(1995); both of
which are hereby incorporated by reference in their entirety). These Rep
proteins act by a
site-specific and strand-specific endonuclease nick at the AAV origin at the
terminal
resolution site, followed by covalent attachment to the 5' terminus of the
nicked site via a
putative tyrosine linkage. Rep68 and Rep78 result from alternate splicing of
the
transcript. The nucleic acid sequence of Rep68 is shown in Figure 15, and the
protein
sequence in Figure 16; the nucleic acid and protein sequences of Rep78
proteins isolated
from various sources are shown in Figures 1, 2, 7, 8, 13, and 14. As is
further outlined
below, functional fragments, variants, and homologs of Rep proteins are also
included
within the definition of Rep proteins; in this case, the variants preferably
include nucleic
acid binding activity and endonuclease activity. The corresponding enzyme
attachment
site for Rep68 and Rep78, discussed below, is shown in Figures 47 and 48 and
is set forth
in Example 1.
In a preferred embodiment, the NAM enzyme is NS 1. NS 1 is a non-structural
protein in parvovirus, is a functional homolog of Rep78, and also covalently
attaches to
DNA (Cotmore et al., J. Virol., 62(3), 851-860 (1998), hereby expressly
incorporated by
reference). The nucleotide and amino acid sequences of NS 1 proteins isolated
from
various sources are shown in Figures 9-12, 29-34, 37, and 38. As is further
outlined
below, fragments and variants of NS 1 proteins are also included within the
definition of
NS 1 proteins.
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In a preferred embodiment, the NAM enzyme is the parvoviral H-1 protein, which
is also known to form a covalent linkage with DNA (see, for example, Tseng et
al., Proc.
Natl. Acad. Sci. USA, 76( 11 ), 5539-5543 ( 1979), hereby expressly
incorporated by
reference. As is further outlined below, fragments and variants of H-1
proteins are also
included within the definition of H-1 proteins.
In a preferred embodiment, the NAM enzyme is the bacteriophage phi-29 terminal
protein, which is also known to form a covalent linkage with DNA (see, for
example,
Germendia et al., Nucleic Acid Research, 16(3), 5727-5740 (1988), hereby
expressly
incorporated by reference). As is further outlined below, fragments and
variants of phi-29
proteins are also included within the definition of phi-29 proteins.
The NAM enzyme also can be the adenoviral 55 Kd (a55) protein, again known to
form covalent linkages with DNA; see Desiderio and Kelly, J. Mol. Biol., 98,
319-337
( 1981 ), hereby expressly incorporated by reference. As is further outlined
below,
fragments and variants of a55 proteins are also included within the definition
of a55
proteins.
The nucleic acid sequences and amino acid sequences of other Rep homologs that
are suitable for use as NAM enzymes are set forth in Figures 3-6, 17-28, 35,
36, and 39-
46.
Some DNA-binding enzymes form covalent linkages upon physical or chemical
stimuli such as, for example, UV-induced crosslinking between DNA and a bound
protein, or camptothecin (CPT)-related chemically induced trapping of the DNA-
topoisomerase I covalent complex (e.g., Hertzberg et al., J. Biol. Chem., 265,
19287-
19295 ( 1990)). NAM enzymes that form induced covalent linkages are suitable
for use in
some embodiments of the present invention.
Also included with the definition of NAM enzymes of the present invention are
amino acid sequence variants retaining biological activity (e.g., the ability
to covalently
attach to nucleic acid molecules). These variants fall into one or more of
three classes:
substitutional, insertional or deletional (e.g. fragment) variants. These
variants ordinarily
are prepared by site specific mutagenesis of nucleotides in the DNA encoding
the NAM
protein, using cassette or PCR mutagenesis or other techniques well known in
the art, to
produce DNA encoding the variant, and thereafter expressing the recombinant
DNA in
cell culture as outlined herein. However, variant NAM protein fragments having
up to
about 100-150 residues may be prepared by in vitro synthesis or peptide
ligation using
established techniques. Amino acid sequence variants are characterized by the
predetermined nature of the variation, a feature that sets them apart from
naturally
occurring allelic or interspecies variation of the NAM protein amino acid
sequence. The
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variants typically exhibit the same qualitative biological activity as the
naturally occurring
analogue, although variants can also be selected which have modified
characteristics as
will be more fully outlined below.
While the site or region for introducing an amino acid sequence variation is
5 predetermined, the mutation per se need not be predetermined. For example,
in order to
optimize the performance of a mutation at a given site, random mutagenesis may
be
conducted at the target codon or region and the expressed NAM variants
screened for the
optimal combination of desired activity. Techniques for making substitution
mutations at
predetermined sites in DNA having a known sequence are well known, for
example, M13
10 primer mutagenesis and PCR mutagenesis. Screening of the mutants, variants,
homologs,
etc., is accomplished using assays of NAM protein activities employing routine
methods
such as, for example, binding assays, affinity assays, peptide conformation
mapping, and
the like.
Amino acid substitutions are typically of single residues; insertions usually
will be
15 on the order of from about 1 to 20 amino acids, although considerably
larger insertions
may be tolerated. Deletions range from about 1 to about 20 residues, although
in some
cases deletions may be much larger, for example when unnecessary domains are
removed.
Substitutions, deletions, insertions or any combination thereof may be used to
arrive at a final derivative. Generally these changes are done on a few amino
acids to
minimize the alteration of the molecule. However, larger changes may be
tolerated in
certain circumstances. When small alterations in the characteristics of the
NAM protein
are desired, substitutions are generally made in accordance with the following
chart:
Chart I
Original Residue Exemplary Substitutions
Ala Ser
Arg Lys
Asn Gln, His
Asp Glu
Cys Ser
Gln Asn
Glu Asp
Gly Pro
His Asn, Gln
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Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
PheSer Met, Leu, Tyr
Thr Thr
Trp Ser
Tyr Tyr
Val Trp, Phe
Ile, Leu
Substantial changes in function or immunological identity are made by
selecting substitutions that are less conservative than those shown in Chart
I. For
example, substitutions may be made which more significantly affect: the
structure
of the polypeptide backbone in the area of the alteration, for example the
alpha-
s helical or beta-sheet structure; the charge or hydrophobicity of the
molecule at the
target site; or the bulk of the side chain. The substitutions which in general
are
expected to produce the greatest changes in the polypeptide's properties are
those
in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for
(or by) a
hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl;
(b) a
cysteine or proline is substituted for (or by) any other residue; (c) a
residue having
an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is
substituted for (or
by) an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue
having a
bulky side chain, e.g. phenylalanine, is substituted for (or by) one not
having a side
chain, e.g. glycine.
The variants typically exhibit the same qualitative biological activity as the
naturally-occurring analogue, although variants also are selected to modify
the
characteristics of the NAM proteins as needed. Alternatively, the variant may
be
designed such that the biological activity of the NAM protein is altered. For
example, glycosylation sites may be altered or removed. Similarly, functional
mutations within the endonuclease domain or nucleic acid recognition site may
be
made. Furthermore, unnecessary domains may be deleted, to form fragments of
NAM enzymes.
In addition, some embodiments utilize concatameric constructs to effect
multivalency and increase binding kinetics or efficiency. For example,
constructs
containing a plurality of NAM coding regions or a plurality of EASs may be
made.
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17
Also included with the definition of NAM protein are other NAM
homologs, and NAM proteins from other organisms including viruses, which are
cloned and expressed as known in the art. Thus, probe or degenerate polymerase
chain reaction (PCR) primer sequences may be used to find other related NAM
proteins. As will be appreciated by those in the art, particularly useful
probe and/or
PCR primer sequences include the unique areas of the NAM nucleic acid
sequence. As is generally known in the art, preferred PCR primers are from
about
to about 35 nucleotides in length, with from about 20 to about 30 being
10 preferred, and may contain inosine as needed. The conditions for the PCR
reaction
are well known in the art.
In addition to nucleic acids encoding NAM enzymes, the fusion nucleic
acids of the invention also encode candidate proteins. By "protein" herein is
meant at least two covalently attached amino acids, which includes proteins,
15 polypeptides, oligopeptides and peptides. The protein may be made up of
naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic
structures, the latter being especially useful when the target molecule is a
protein.
Thus "amino acid", or "peptide residue", as used herein means both naturally
occurring and synthetic amino acids. For example, homo-phenylalanine,
citrulline
and noreleucine are considered amino acids for the purposes of the invention.
"Amino acid" also includes imino acid residues such as proline and
hydroxyproline. The side chains may be in either the (R) or the (S)
configuration.
In the preferred embodiment, the amino acids are in the (S) or L-
configuration. If
non-naturally occurring side chains are used, non-amino acid substituents may
be
used, for example to prevent or retard ex vivo degradations. Chemical blocking
groups or other chemical substituents may also be added. Thus, the present
invention can find use in template based synthetic systems.
By "candidate protein" herein is meant a protein to be tested for binding,
association or effect in an assay of the invention, including both in vitro
(e.g. cell
free systems) or ex vivo (within cells). The candidate peptide comprises at
least
one desired target property. The desired target property will depend upon the
particular embodiment of the present invention. "Target property" refers to an
activity of interest. Optionally, the target property is used directly or
indirectly to
identify a subset of fusion protein-expression vector conjugates, thus
allowing for
the retrieval of the desired NAP conjugates from the fusion protein library.
Target
properties include, for example, the ability of the encoded display peptide to
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18
mediate binding to a partner, enzymatic activity, the ability to mimic a given
factor, the ability to alter cell physiology, and structural or other physical
properties including, but not limited to, electromagnetic behavior or
spectroscopic
behavior of the peptides. Generally, as outlined below, libraries of candidate
proteins are used in the fusions. As will be appreciated by those in the art,
the
source of the candidate protein libraries can vary, particularly depending on
the
end use of the system.
In a preferred embodiment, the candidate proteins are derived from cDNA
libraries. The cDNA libraries can be derived from any number of different
cells,
particularly those outlined for host cells herein, and include cDNA libraries
generated from eucaryotic and procaryotic cells, viruses, cells infected with
viruses or other pathogens, genetically altered cells, etc. Preferred
embodiments,
as outlined below, include cDNA libraries made from different individuals,
such as
different patients, particularly human patients. The cDNA libraries may be
complete libraries or partial libraries. Furthermore, the library of candidate
proteins can be derived from a single cDNA source or multiple sources; that
is,
cDNA from multiple cell types or multiple individuals or multiple pathogens
can
be combined in a screen. The cDNA library may utilize entire cDNA constructs
or
fractionated constructs, including random or targeted fractionation. Suitable
fractionation techniques include enzymatic, chemical or mechanical
fractionation.
In a preferred embodiment, the candidate proteins are derived from
genomic libraries. As above, the genomic libraries can be derived from any
number of different cells, particularly those outlined for host cells herein,
and
include genomic libraries generated from eucaryotic and procaryotic cells,
viruses,
cells infected with viruses or other pathogens, genetically altered cells,
etc.
Preferred embodiments, as outlined below, include genomic libraries made from
different individuals, such as different patients, particularly human
patients. The
genomic libraries may be complete libraries or partial libraries. Furthermore,
the
library of candidate proteins can be derived from a single genomic source or
multiple sources; that is, genomic DNA from multiple cell types or multiple
individuals or multiple pathogens can be combined in a screen. The genomic
library may utilize entire genomic constructs or fractionated constructs,
including
random or targeted fractionation. Suitable fractionation techniques include
enzymatic, chemical or mechanical fractionation.
In this regard, the combination of a NAM enzyme with nucleic acid derived
from genomic DNA in a genetic library vector is novel. Accordingly, the
present
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19
invention further provides an isolated and purified nucleic acid molecule
comprising a nucleic acid sequence encoding a NAM enzyme fused to a nucleic
acid sequence isolated from genomic DNA. Such an isolated and purified nucleic
acid molecule is particularly useful in the present inventive methods
described
herein. Preferably, the isolated and purified nucleic acid molecule further
comprises a splice donor sequence or splice acceptor sequence located between
the
nucleic acid sequence encoding the NAM enzyme and the genomic DNA. The
incorporation of splice donor and/or splice acceptor sequences into the
isolated
and purified nucleic acid sequence allows formation of a transcript encoding
the
NAM enzyme and exons of the genomic DNA fragment. The methods of the prior
art have failed to comprehend the potential of operably linking genomic DNA to
a
NAM enzyme such that the product of the genomic DNA can be associated with
the nucleic acid molecule encoding it. One of ordinary skill in the art will
appreciate that appropriate regulatory sequences can also be incorporated into
the
isolated and purified nucleic acid molecule.
In a preferred embodiment, the present invention also provides methods of
determining open reading frames in genomic DNA. In this embodiment, the
candidate protein encoded by the genomic nucleic acid is preferably fused
directly
to the N-terminus of the NAM enzyme, rather than at the C-terminus. Thus, if a
functional NAM enzyme is produced, the genomic DNA was fused in the correct
reading frame. This is particularly useful with the use of labels, as well.
In addition, the libraries may also be subsequently mutated using known
techniques (exposure to mutagens, error-prone PCR, error-prone transcription,
combinatorial splicing (e.g. cre-lox recombination)). In this way libraries of
procaryotic and eukaryotic proteins may be made for screening in the systems
described herein. Particularly preferred in this embodiment are libraries of
bacterial, fungal, viral, plant, and animal (e.g., mammalian) proteins, with
the
latter being preferred, and human proteins being especially preferred.
The candidate proteins may vary in size. In the case of cDNA or genomic
libraries, the proteins may range from 20 or 30 amino acids to thousands, with
from about 50 to 1000 (e.g., 75, 150, 350, 750 or more) being preferred and
from
100 to 500 (e.g., 200, 300, or 400) being especially preferred. When the
candidate
proteins are peptides, the peptides are from about 3 to about 50 amino acids,
with
from about 5 to about 20 amino acids being preferred, and from about 7 to
about
1 S being particularly preferred. The peptides may be digests of naturally
occurring
proteins as is outlined above, random peptides, or "biased" random peptides.
By
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"randomized" or grammatical equivalents herein is meant that each nucleic acid
and peptide consists of essentially random nucleotides and amino acids,
respectively. Since generally these random peptides (or nucleic acids,
discussed
below) are chemically synthesized, they may incorporate any nucleotide or
amino
5 acid at any position. The synthetic process can be designed to generate
randomized proteins or nucleic acids, to allow the formation of all or most of
the
possible combinations over the length of the sequence, thus forming a library
of
randomized candidate bioactive proteinaceous agents.
In a preferred embodiment, libraries of candidate proteins are fused to the
10 NAM enzymes, with each member of the library comprising a different
candidate
protein. However, as will be appreciated by those in the art, different
members of
the library may be reproduced or duplicated, resulting in some libraries
members
being identical. The library should provide a sufficiently structurally
diverse
population of expression products to effect a probabilistically sufficient
range of
1 S cellular responses to provide one or more cells exhibiting a desired
response.
Accordingly, an interaction library must be large enough so that at least one
of its
members will have a structure that gives it affinity for some molecule,
including
both protein and non-protein targets, or other factors whose activity is
necessary or
effective within the assay of interest. Although it can be difficult to gauge
the
20 required absolute size of an interaction library, nature provides a hint
with the
immune response: a diversity of 10'-10g different antibodies provides at least
one
combination with sufficient affinity to interact with most potential antigens
faced
by an organism. Published in vitro selection techniques have also shown that a
library size of 10' to 108 is sufficient to find structures with affinity for
the target.
A library of all combinations of a peptide 7 to 20 amino acids in length has
the
potential to code for 20' ( 109) to 202° . Thus, with libraries of 10'
to 1 Og the
present methods allow a "working" subset of a theoretically complete
interaction
library for 7 amino acids, and a subset of shapes for the 202° library.
Thus, in a
preferred embodiment, at least 106, preferably at least 10', more preferably
at least
108 and most preferably at least 109 different expression products are
simultaneously analyzed in the subject methods, although libraries of less
complexity (e.g., 102, 103, 104, or 105 different expression products) or
greater
complexity (e.g., 10'°, 10'', or 10'Z different expression products)
are appropriate
for use in the present invention. Preferred methods maximize library size and
diversity.
In any library system encoded by oligonucleotide synthesis, complete
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21
control over the codons that will eventually be incorporated into the peptide
structure is difficult. This is especially true in the case of codons encoding
stop
signals (TAA, TGA, TAG). In a synthesis with NNN as the random region, there
is a 3/64, or 4.69%, chance that the codon will be a stop codon. Thus, in a
peptide
of 10 residues, there is a high likelihood that 46.7% of the peptides will
prematurely terminate. One way to alleviate this is to have random residues
encoded as NNK, where K= T or G. This allows for encoding of all potential
amino acids (changing their relative representation slightly), but importantly
preventing the encoding of two stop residues TAA and TGA. Thus, libraries
encoding a 10 amino acid peptide will have a 15.6% chance to terminate
prematurely. Alternatively, fusing the candidate proteins to the C-terminus of
the
NAM enzyme also may be done, although in some instances, fusing to the N-
terminus means that prematurely terminating proteins result in a lack of NAM
enzyme which eliminates these samples from the assay.
In one embodiment, the library is fully randomized, with no sequence
preferences or constants at any position. In a preferred embodiment, the
library is
biased. That is, some positions within the sequence are either held constant,
or are
selected from a limited number of possibilities. For example, in a preferred
embodiment, the nucleotides or amino acid residues are randomized within a
defined class, for example, of hydrophobic amino acids, hydrophilic residues,
sterically biased (either small or large) residues, towards the creation of
cysteines,
for cross-linking, prolines for SH-3 domains, PDZ domains, serines,
threonines,
tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
In a preferred embodiment, the bias is towards peptides or nucleic acids
that interact with known classes of molecules. For example, when the candidate
protein is a peptide, it is known that much'of intracellular signaling is
carried out
via short regions of polypeptides interacting with other polypeptides through
small
peptide domains. For instance, a short region from the HIV-1 envelope
cytoplasmic domain has been previously shown to block the action of cellular
calmodulin. Regions of the Fas cytoplasmic domain, which shows homology to
the mastoparan toxin from Wasps, can be limited to a short peptide region with
death-inducing apoptotic or G protein inducing functions. Magainin, a natural
peptide derived from Xenopus, can have potent anti-tumour and anti-microbial
activity. Short peptide fragments of a protein kinase C isozyme (13PKC), have
been shown to block nuclear translocation of 13PKC in Xenopus oocytes
following
stimulation. And, short SH-3 target peptides have been used as
pseudosubstrates
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22
for specific binding to SH-3 proteins. This is of course a short list of
available
peptides with biological activity, as the literature is dense in this area.
Thus, there
is much precedent for the potential of small peptides to have activity on
intracellular signaling cascades. In addition, agonists and antagonists of any
number of molecules may be used as the basis of biased randomization of
candidate proteins as well.
Thus, a number of molecules or protein domains are suitable as starting
points for the generation of biased randomized candidate proteins. A large
number
of small molecule domains are known, that confer a common function, structure
or
affinity. In addition, as is appreciated in the art, areas of weak amino acid
homology may have strong structural homology. A number of these molecules,
domains, and/or corresponding consensus sequences, are known, including, but
are
not limited to, SH-2 domains, SH-3 domains, Pleckstrin, death domains,
protease
cleavage/recognition sites, enzyme inhibitors, enzyme substrates, Traf, etc.
Similarly, there are a number of known nucleic acid binding proteins
containing
domains suitable for use in the invention. For example, leucine zipper
consensus
sequences are known.
In a preferred embodiment, biased SH-3 domain-binding
oligonucleotides/peptides are made. SH-3 domains have been shown to recognize
short target motifs (SH-3 domain-binding peptides), about ten to twelve
residues in
a linear sequence, that can be encoded as short peptides with high affinity
for the
target SH-3 domain. Consensus sequences for SH-3 domain binding proteins have
been proposed. Thus, in a preferred embodiment, oligos/peptides are made with
the following biases:
1. XXXPPXPXX, wherein X is a randomized residue.
2. (within the positions of residue positions 11 to -2):
11 10 9 8 7 6 5 4 3 2 1
Met Gly aal l aal0 aa9 aa8 aa7 Arg Pro Leu Pro Pro hyd
0 -1 -2
Pro hyd hyd Gly Gly Pro Pro STOP
atg ggc nnk nnk nnk nnk nnk aga cct ctg cct cca sbk ggg sbk sbk gga ggc cca
cct
TAA 1.
In this embodiment, the N-terminus flanking region is suggested to have
the greatest effects on binding affinity and is therefore entirely randomized.
"Hyd" indicates a bias toward a hydrophobic residue, i.e.- Val, Ala, Gly, Leu,
Pro,
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23
Arg. To encode a hydrophobically biased residue, "sbk" codon biased structure
is
used. Examination of the codons within the genetic code will ensure this
encodes
generally hydrophobic residues. s= g,c; b= t, g, c; v= a, g, c; m= a, c; k= t,
g; n= a,
t, g, c.
Thus, in a preferred embodiment, the candidate protein is a structural tag
that will allow the isolation of target proteins with that structure. That is,
in the
case of leucine zippers, the fusion of the NAM enzyme to a leucine zipper
sequence will allow the fusions to "zip up" with other leucine zippers, allow
the
quick isolation of a plurality of leucine zipper proteins. In addition,
structural tags
(which may only be the proteins themselves) can allow heteromultimeric protein
complexes to form, that then are assayed for activity as complexes. That is,
many
proteins, such as many eucaryotic transcription factors, function as
heteromultimeric complexes which can be assayed using the present invention.
In addition, rather than a cDNA, genomic, or random library, the candidate
1 S protein library may be a constructed library; that is, it may be built to
contain only
members of a defined class, or combinations of classes. For example, libraries
of
immunoglobulins may be built, or libraries of G-protein coupled receptors,
tumor
suppressor genes, proteases, transcription factors, phosphatases, kinases,
etc.
The fusion nucleic acid can comprise the NAM enzyme and candidate
protein in a variety of configurations, including both direct and indirect
fusions,
and include N- and C-terminal fusions and internal fusions.
In a preferred embodiment, the NAM enzyme and the candidate protein are
directly fused. In this embodiment, a direct, in-frame fusion of the nucleic
acid
encoding the NAM enzyme and the candidate protein is engineered. The library
of
fusion peptides can be constructed as N- and/or C-terminal fusions and
internal
fusions. Thus, the NAM enzyme coding region may be 3' or 5' to the candidate
protein coding region, or the candidate protein coding region may be inserted
into
a suitable position within the coding region of the NAM enzyme. In this
embodiment, it may be desirable to insert the candidate protein into an
external
loop of the NAM enzyme, either as a direct insertion or with the replacement
of
several of the NAM enzyme residues. This may be particularly desirable in the
case of random candidate proteins, as they frequently require some sort of
scaffold
or presentation structure to confer a conformationally restricted structure.
For an
example of this general idea using green fluorescent protein (GFP) as a
scaffold
for the expression of random peptide libraries, see for example WO 99/20574,
expressly incorporated herein by reference.
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In a preferred embodiment, the NAM enzyme and the candidate protein are
indirectly fused. This may be accomplished such that the components of the
fusion remain attached, such as through the use of linkers, or in ways that
result in
the components of the fusion becoming separated. As will be appreciated by
those
in the art, there are a wide variety of different types of linkers that may be
used,
including cleavable and non-cleavable linkers; this cleavage may also occur at
the
level of the nucleic acid, or at the protein level.
In a preferred embodiment, linkers may be used to functionally isolate the
NAM enzyme and the candidate protein. That is, a direct fusion system may
sterically or functionally hinder the interaction of the candidate protein
with its
intended binding partner, and thus fusion configurations that allow greater
degrees
of freedom are useful. An analogy is seen in the single chain antibody area,
where
the incorporation of a linker allows functionality.
In a preferred embodiment, linkers known to confer flexibility are used.
For example, useful linkers include glycine-serine polymers (including, for
example, (GS)~, and (GGGS)", where n is an integer of at least one), glycine-
alanine polymers, alanine-serine polymers, and other flexible linkers such as
the
tether for the shaker potassium channel, and a large variety of other flexible
linkers, as will be appreciated by those in the art. Glycine-serine polymers
are
preferred since both of these amino acids are relatively unstructured, and
therefore
may be able to serve as a neutral tether between components. Secondly, serine
is
hydrophilic and therefore able to solubilize what could be a globular glycine
chain.
Third, similar chains have been shown to be effective in joining subunits of
recombinant proteins such as single chain antibodies.
The linker used to construct indirect fusion enzymes can be a cleavable
linker. Cleavable linkers can function at the level of the nucleic acid or the
protein.
That is, cleavage (which in this sense means that the NAM enzyme and the
candidate protein are separated) can occur during transcription, or before or
after
translation.
With respect to cleavable linkers, the cleavage can occur as a result of a
cleavage functionality built into the nucleic acid. In this embodiment, for
example, cleavable nucleic acid sequences, or sequences that will disrupt the
nucleic acid, can be used. For example, intron sequences that the cell will
remove
can be placed between the coding region of the NAM enzyme and the candidate
protein. In a preferred embodiment, the linkers are heterodimerization
domains. In
this embodiment, both the NAM enzyme and the candidate protein are fused to
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heterodimerization domains (or multimeric domains, if multivalency is
desired), to
allow association of these two proteins after translation.
In a preferred embodiment, cleavable protein linkers are used. In this
embodiment, the fusion nucleic acids include coding sequences for a protein
5 sequence that may be subsequently cleaved, generally by a protease. As will
be
appreciated by those in the art, cleavage sites directed to ubiquitous
proteases, e.g.
those that are constitutively present in most or all of the host cells of the
system,
can be used. Alternatively, cleavage sites that correspond to cell-specific
proteases
may be used. Similarly, cleavage sites for proteases that are induced only
during
10 certain cell cycles or phases or are signal specific events may be used as
well.
There are a wide variety of possible proteinaceous cleavage sites known.
For example, sequences that are recognized and cleaved by a protease or
cleaved
after exposure to certain chemicals are considered cleavable linkers. This may
find particular use in in vitro systems, outlined below, as exogeneous enzymes
can
15 be added to the milieu or the NAP conjugates may be purified and the
cleavage
agents added. For example, cleavable linkers include, but are not limited to,
the
prosequence of bovine chymosin, the prosequence of subtilisin, the 2a site
(Ryan
et al., J. Gen. Virol. 72:2727 ( 1991 ); Ryan et al., EMBO J. 13:928 ( 1994);
Donnelly et al., J. Gen. Virol. 78:13 (1997); Hellen et al., Biochem,
28(26):9881
20 ( 1989); and Mattion et al., J. Virol. 70:8124 ( 1996)), prosequences of
retroviral
proteases including human immunodeficiency virus protease and sequences
recognized and cleaved by trypsin (EP 578472, Takasuga et al., J. Biochem.
112(5)652 (1992)) factor Xa (Gardella et al., J. Biol. Chem. 265(26):15854
(1990),
WO 9006370), collagenase (J03280893, Tajima et al., J. Ferment. Bioeng.
25 72(5):362 (1991), WO 9006370), clostripain (EP 578472), subtilisin
(including
mutant H64A subtilisin, Forsberg et al., J. Protein Chem. 10(5):517 ( 1991 ),
chymosin, yeast KEX2 protease (Bourbonnais et al., J. Bio. Chem. 263(30):15342
( 1988), thrombin (Forsberg et al., supra; Abath et al., BioTechniques
10(2):178
(1991)), Staphylococcus aureus V8 protease or similar endoproteinase-Glu-C to
cleave after Glu residues (EP 578472, Ishizaki et al., Appl. Microbiol.
Biotechnol.
36(4):483 (1992)), cleavage by NIa proteainase of tobacco etch virus (Parks et
al.,
Anal. Biochem. 216(2):413 (1994)), endoproteinase-Lys-C (U.S. Patent No.
4,414,332) and endoproteinase-Asp-N, Neisseria type 2 IgA protease (Pohlner et
al., Bio/Technology 10(7):799-804 (1992)), soluble yeast endoproteinase yscF
(EP
467839), chymotrypsin (Altman et al., Protein Eng. 4(5):593 (1991)),
enteropeptidase (WO 9006370), lysostaphin, a polyglycine specific
endoproteinase
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26
(EP 316748), and the like. See e.g. Marston, F.A.O. (1986) Biol. Chem. J. 240,
1-12. Particular amino acid sites that serve as chemical cleavage sites
include, but
are not limited to, methionine for cleavage by cyanogen bromide (Shen, PNAS
USA 81:4627 (1984); Kempe et al., Gene 39:239 (1985); Kuliopulos et al., J.
Am.
Chem. Soc. 116:4599 ( 1994); Moks et al., Bio/Technology 5:379 ( 1987); Ray et
al., Bio/Technology 11:64 (1993)), acid cleavage of an Asp-Pro bond (Wingender
et al., J. Biol. Chem. 264(8):4367 (1989); Gram et al., Bio/Technology 12:1017
( 1994)), and hydroxylamine cleavage at an Asn-Gly bond (Moks, supra).
In addition to the NAM enzymes, candidate proteins, and linkers, the fusion
nucleic acids can comprise additional coding sequences for other
functionalities.
As will be appreciated by those in the art, the discussion herein is directed
to
fusions of these other components to the fusion nucleic acids described
herein;
however, they can also be separate from the fusion protein and rather be a
component of the expression vector comprising the fusion nucleic acid, as is
generally outlined below.
Thus, in a preferred embodiment, the fusions are linked to a fusion partner.
By "fusion partner" or "functional group" herein is meant a sequence that is
associated with the candidate protein, that confers upon all members of the
library
in that class a common function or ability. Fusion partners can be
heterologous
(i.e. not native to the host cell), or synthetic (not native to any cell).
Suitable fusion
partners include, but are not limited to: a) presentation structures, as
defined
below, which provide the candidate proteins in a conformationally restricted
or
stable form, including hetero- or homodimerization or multimerization
sequences;
b) targeting sequences, defined below, which allow the localization of the
candidate proteins into a subcellular or extracellular compartment or be
incorporated into infected organisms, such as those infected by viruses or
pathogens; c) rescue sequences as defined below, which allow the purification
or
isolation of the NAP conjugates; d) stability sequences, which confer
stability or
protection from degradation to the candidate protein or the nucleic acid
encoding
it, for example resistance to proteolytic degradation; e) linker sequences; or
f) any
combination of a), b), c), d), and e), as well as linker sequences as needed.
In a preferred embodiment, the fusion partner is a presentation structure.
By "presentation structure" or grammatical equivalents herein is meant an
amino
acid sequence, which, when fused to candidate proteins, causes the candidate
proteins to assume a conformationally restricted form. This is particularly
useful
when the candidate proteins are random, biased random or pseudorandom
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27
peptides. Proteins interact with each other largely through conformationally
constrained domains. Although small peptides with freely rotating amino and
carboxyl termini can have potent functions as is known in the art, the
conversion
of such peptide structures into pharmacologic agents is difficult due to the
inability
to predict side-chain positions for peptidomimetic synthesis. Therefore the
presentation of peptides in conformationally constrained structures will
benefit
both the later generation of pharmaceuticals and will also likely lead to
higher
affinity interactions of the peptide with the target protein. This fact has
been
recognized in the combinatorial library generation systems using biologically
generated short peptides in bacterial phage systems.
Thus, synthetic presentation structures, i.e. artificial polypeptides, are
capable of presenting a randomized peptide as a conformationally-restricted
domain. Generally such presentation structures comprise a first portion joined
to
the N-terminal end of the randomized peptide, and a second portion joined to
the
C-terminal end of the peptide; that is, the peptide is inserted into the
presentation
structure, although variations may be made, as outlined below. To increase the
functional isolation of the randomized expression product, the presentation
structures are selected or designed to have minimal biologically activity when
expressed in the target cell.
Preferred presentation structures maximize accessibility to the peptide by
presenting it on an exterior loop. Accordingly, suitable presentation
structures
include, but are not limited to, minibody structures, dimerization sequences,
loops
on beta-sheet turns and coiled-coil stem structures in which residues not
critical to
structure are randomized, zinc-finger domains, cysteine-linked (disulfide)
structures, transglutaminase linked structures, cyclic peptides, B-loop
structures,
helical barrels or bundles, leucine zipper motifs, etc.
In a preferred embodiment, the presentation structure is a coiled-coil
structure, allowing the presentation of the randomized peptide on an exterior
loop.
See, for example, Myszka et al., Biochem. 33:2362-2373 ( 1994), hereby
incorporated by reference, and Figure 3). Using this system investigators have
isolated peptides capable of high affinity interaction with the appropriate
target. In
general, coiled-coil structures allow for between 6 to 20 randomized
positions. A
preferred coiled-coil presentation structure is described in, for example,
Martin et
al., EMBO J. 13(22):5303-5309 (1994), incorporated by reference.
In a preferred embodiment, the presentation structure is a minibody
structure. A "minibody" is essentially composed of a minimal antibody
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28
complementarity region. The minibody presentation structure generally provides
two randomizing regions that in the folded protein are presented along a
single
face of the tertiary structure. See, for example, Bianchi et al., J. Mol.
Biol.
236(2):649-59 ( 1994), and references cited therein, all of which are
incorporated
by reference. Investigators have shown this minimal domain is stable in
solution
and have used phage selection systems in combinatorial libraries to select
minibodies with peptide regions exhibiting high affinity, Kd = 10-', for the
pro-
inflammatory cytokine IL-6.
A preferred minibody presentation structure is as follows:
MGRNSQATSGFTFSHFYMEWVRGGEYIAASRHKHNKYTTEYSASVKGR
YIVSRDTSQSILYLQKKKGPP (SEQ ID NO:1). The bold, underline regions are
the regions which may be randomized. The italized phenylalanine must be
invariant in the first randomizing region. The entire peptide is cloned in a
three-
oligonucleotide variation of the coiled-coil embodiment, thus allowing two
different randomizing regions to be incorporated simultaneously. This
embodiment utilizes non-palindromic BstXI sites on the termini.
In a preferred embodiment, the presentation structure is a sequence that
contains generally two cysteine residues, such that a disulfide bond may be
formed, resulting in a conformationally constrained sequence. This embodiment
is
particularly preferred when secretory targeting sequences are used. As will be
appreciated by those in the art, any number of random sequences, with or
without
spacer or linking sequences, may be flanked with cysteine residues. In other
embodiments, effective presentation structures may be generated by the random
regions themselves. For example, the random regions may be "doped" with
cysteine residues which, under the appropriate redox conditions, may result in
highly crosslinked structured conformations, similar to a presentation
structure.
Similarly, the randomization regions may be controlled to contain a certain
number of residues to confer 13-sheet or a-helical structures.
In one embodiment, the presentation structure is a dimerization or
multimerization sequence. A dimerization sequence allows the non-covalent
association of one candidate protein to another candidate protein, including
peptides, with sufficient affinity to remain associated under normal
physiological
conditions. This effectively allows small libraries of candidate protein (for
example, 104) to become large libraries if two proteins per cell are generated
which then dimerize, to form an effective library of 10g (104 X 104). It also
allows
the formation of longer proteins, if needed, or more structurally complex
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29
molecules. The dimers may be homo- or heterodimers.
Dimerization sequences may be a single sequence that self aggregates, or
two sequences. That is, nucleic acids encoding both a first candidate protein
with
dimerization sequence 1, and a second candidate protein with dimerization
sequence 2, such that upon introduction into a cell and expression of the
nucleic
acid, dimerization sequence 1 associates with dimerization sequence 2 to form
a
new structure.
Suitable dimerization sequences will encompass a wide variety of
sequences. Any number of protein-protein interaction sites are known. In
addition, dimerization sequences may also be elucidated using standard methods
such as the yeast two hybrid system, traditional biochemical affinity binding
studies, or even using the present methods.
In a preferred embodiment, the fusion partner is a targeting sequence. As
will be appreciated by those in the art, the localization of proteins within a
cell is a
simple method for increasing effective concentration and determining function.
For example, RAF 1 when localized to the mitochondria) membrane can inhibit
the
anti-apoptotic effect of BCL-2. Similarly, membrane bound Sos induces Ras
mediated signaling in T-lymphocytes. These mechanisms are thought to rely on
the
principle of limiting the search space for ligands, that is to say, the
localization of a
protein to the plasma membrane limits the search for its ligand to that
limited
dimensional space near the membrane as opposed to the three dimensional space
of the cytoplasm. Alternatively, the concentration of a protein can also be
simply
increased by nature of the localization. Shuttling the proteins into the
nucleus
confines them to a smaller space thereby increasing concentration. Finally,
the
ligand or target may simply be localized to a specific compartment, and
inhibitors
must be localized appropriately.
Thus, suitable targeting sequences include, but are not limited to, binding
sequences capable of causing binding of the expression product to a
predetermined
molecule or class of molecules while retaining bioactivity of the expression
product, (for example by using enzyme inhibitor or substrate sequences to
target a
class of relevant enzymes); sequences signaling selective degradation, of
itself or
co-bound proteins; and signal sequences capable of constitutively localizing
the
candidate expression products to a predetermined cellular locale, including a)
subcellular locations such as the Golgi, endoplasmic reticulum, nucleus,
nucleoli,
nuclear membrane, mitochondria, chloroplast, secretory vesicles, lysosome, and
cellular membrane or within pathogens or viruses that have infected the cell;
and
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b) extracellular locations via a secretory signal. Particularly preferred is
localization to either subcellular locations or to the outside of the cell via
secretion.
In a preferred embodiment, the targeting sequence is a nuclear localization
5 signal (NLS). NLSs are generally short, positively charged (basic) domains
that
serve to direct the entire protein in which they occur to the cell's nucleus.
Numerous NLS amino acid sequences have been reported including single basic
NLS's such as that of the SV40 (monkey virus) large T Antigen (Pro Lys Lys Lys
Arg Lys Val), Kalderon (1984), et al., Cell, 39:499-509; the human retinoic
acid
10 receptor-13 nuclear localization signal; NFkB p50 (see, for example, Ghosh
et al.,
Cell 62:1019 ( 1990)); NFkB p65 (see, for example, Nolan et al., Cell 64:961
( 1991 )); and others (see, for example, Boulikas, J. Cell. Biochem. 55( 1
):32-58
( 1994), hereby incorporated by reference) and double basic NLS's exemplified
by
that of the Xenopus (African clawed toad) protein, nucleoplasmin (see, for
15 example, Dingwall, et al., Cell, 30:449-458, 1982 and Dingwall, et al., J.
Cell
Biol., 107:641-849; 1988). Numerous localization studies have demonstrated
that
NLSs incorporated in synthetic peptides or grafted onto reporter proteins not
normally targeted to the cell nucleus cause these peptides and reporter
proteins to
be concentrated in the nucleus. See, for example, Dingwall, and Laskey, Ann,
20 Rev. Cell Biol., 2:367-390, 1986; Bonnerot, et al., Proc. Natl. Acad. Sci.
USA,
84:6795-6799, 1987; Galileo, et al., Proc. Natl. Acad. Sci. USA, 87:458-462,
1990.
In a preferred embodiment, the targeting sequence is a membrane
anchoring signal sequence. This is particularly useful since many parasites
and
25 pathogens bind to the membrane, in addition to the fact that many
intracellular
events originate at the plasma membrane. Thus, membrane-bound peptide
libraries are useful for both the identification of important elements in
these
processes as well as for the discovery of effective inhibitors. In addition,
many
drugs interact with membrane associated proteins. The invention provides
30 methods for presenting the candidate proteins extracellularly or in the
cytoplasmic
space. For extracellular presentation, a membrane anchoring region is provided
at
the carboxyl terminus of the candidate protein. The candidate protein region
is
expressed on the cell surface and presented to the extracellular space, such
that it
can bind to other surface molecules (affecting their function) or molecules
present
in the extracellular medium. The binding of such molecules could confer
function
on the cells expressing a peptide that binds the molecule. The cytoplasmic
region
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31
could be neutral or could contain a domain that, when the extracellular
candidate
protein region is bound, confers a function on the cells (activation of a
kinase,
phosphatase, binding of other cellular components to effect function).
Similarly,
the candidate protein-containing region could be contained within a
cytoplasmic
region, and the transmembrane region and extracellular region remain constant
or
have a defined function.
In addition, it should be noted that in this embodiment, as well as others
outlined herein, it is possible that the formation of the NAP conjugate
happens
after the screening; that is, having the fusion protein expressed on the
extracellular
surface means that it may not be available for binding to the nucleic acid.
However, this may be done later, with lysis of the cell.
Membrane-anchoring sequences are well known in the art and are based on
the genetic geometry of mammalian transmembrane molecules. Peptides are
inserted into the membrane based on a signal sequence (designated herein as
ssTM) and require a hydrophobic transmembrane domain (herein TM). The
transmembrane proteins are inserted into the membrane such that the regions
encoded 5' of the transmembrane domain are extracellular and the sequences 3'
become intracellular. Of course, if these transmembrane domains are placed 5'
of
the variable region, they will serve to anchor it as an intracellular domain,
which
may be desirable in some embodiments. ssTMs and TMs are known for a wide
variety of membrane bound proteins, and these sequences may be used
accordingly, either as pairs from a particular protein or with each component
being
taken from a different protein, or alternatively, the sequences may be
synthetic,
and derived entirely from consensus as artificial delivery domains.
Membrane-anchoring sequences, including both ssTM and TM, are known
for a wide variety of proteins and any of these may be used. Particularly
preferred
membrane-anchoring sequences include, but are not limited to, those derived
from
CDB, ICAM-2, IL-8R, CD4 and LFA-1.
Useful membrane-anchoring sequences include, for example, sequences
from: 1 ) class I integral membrane proteins such as IL-2 receptor beta-chain
(residues 1-26 are the signal sequence, 241-265 are the transmembrane
residues;
see Hatakeyama et al., Science 244:551 ( 1989) and von Heijne et al, Eur. J.
Biochem. 174:671 (1988)) and insulin receptor beta chain (residues 1-27 are
the
signal, 957-959 are the transmembrane domain and 960-1382 are the cytoplasmic
domain; see Hatakeyama, supra, and Ebina et al., Cell 40:747 ( 1985)); 2)
class II
integral membrane proteins such as neutral endopeptidase (residues 29-51 are
the
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32
transmembrane domain, 2-28 are the cytoplasmic domain; see Malfroy et al.,
Biochem. Biophys. Res. Commun. 144:59 (1987)); 3) type III proteins such as
human cytochrome P450 NF25 (Hatakeyama, supra); and 4) type IV proteins such
as human P-glycoprotein (Hatakeyama, supra). Particularly preferred are CD8
and
ICAM-2. For example, the signal sequences from CD8 and ICAM-2 lie at the
extreme 5' end of the transcript. These consist of the amino acids 1-32 in the
case
of CD8 (see, for example, Nakauchi et al., PNAS USA 82:5126 (1985) and 1-21 in
the case of ICAM-2 (see, for example, Staunton et al., Nature (London) 339:61
( 1989)). These leader sequences deliver the construct to the membrane while
the
hydrophobic transmembrane domains, placed 3' of the random candidate region,
serve to anchor the construct in the membrane. These transmembrane domains are
encompassed by amino acids 145-195 from CD8 (Nakauchi, supra) and 224-256
from ICAM-2 (Staunton, supra).
Alternatively, membrane anchoring sequences can include the GPI anchor,
which results in a covalent bond between the molecule and the lipid bilayer
via a
glycosyl-phosphatidylinositol bond for example in DAF (see, for example,
Homans et al., Nature 333(6170):269-72 (1988), and Moran et al., J. Biol.
Chem.
266:1250 ( 1991 )). In order to do this, the GPI sequence from Thy-1 can be
inserted 3' of the variable region in place of a transmembrane sequence.
Similarly, myristylation sequences can serve as membrane anchoring
sequences. It is known that the myristylation of c-src recruits it to the
plasma
membrane. This is a simple and effective method of membrane localization,
given
that the first 14 amino acids of the protein are solely responsible for this
function
(see Cross et al., Mol. Cell. Biol. 4(9):1834 ( 1984); Spencer et al., Science
262:1019-1024 (1993), both of which are hereby incorporated by reference).
This
motif has already been shown to be effective in the localization of reporter
genes
and can be used to anchor the zeta chain of the TCR. This motif is placed 5'
of the
variable region in order to localize the construct to the plasma membrane.
Other
modifications such as palmitoylation can be used to anchor constructs in the
plasma membrane; for example, palmitoylation sequences from the G protein-
coupled receptor kinase GRK6 sequence (see, for example, Stoffel et al., J.
Biol.
Chem 269:27791 ( 1994)); from rhodopsin (see, for example, Barnstable et al.,
J.
Mol. Neurosci. 5(3):207 (1994)); and the p21 H-ras 1 protein (see, for
example,
Capon et al., Nature 302:33 (1983)).
In a preferred embodiment, the targeting sequence is a lysozomal targeting
sequence, including, for example, a lysosomal degradation sequence such as
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33
Lamp-2 (KFERQ; Dice, Ann. N.Y. Acad. Sci. 674:58 (1992); or lysosomal
membrane sequences from Lamp-1 (see, for example, Uthayakumar et al., Cell.
Mol. Biol. Res. 41:405 ( 1995)) or Lamp-2 (see, for example, Konecki et la.,
Biochem. Biophys. Res. Comm. 205:1-5 (1994)).
Alternatively, the targeting sequence can comprise a mitrochondrial
localization sequence, including mitochondrial matrix sequences (e.g. yeast
alcohol dehydrogenase III; Schatz, Eur. J. Biochem. 165:1-6 (1987));
mitochondrial inner membrane sequences (yeast cytochrome c oxidase subunit IV;
Schatz, supra); mitochondrial intermembrane space sequences (yeast cytochrome
c1; Schatz, supra) or mitochondrial outer membrane sequences (yeast 70 kD
outer
membrane protein; Schatz, supra).
The target sequences also can comprise endoplasmic reticulum sequences,
including the sequences from calreticulin (Pelham, Royal Society London
Transactions B; 1-10 (1992)) or adenovirus E3/19K protein (see, for example,
1 S Jackson et al., EMBO J. 9:3153 ( 1990)).
Furthermore, targeting sequences also can include peroxisome sequences
(for example, the peroxisome matrix sequence from Luciferase; Keller et al.,
PNAS USA 4:3264 (1987)); farnesylation sequences (for example, P21 H-ras 1;
Capon, supra); geranylgeranylation sequences (for example, protein rab-5A;
Farnsworth, PNAS USA 91:11963 ( 1994)); or destruction sequences (cyclin B 1;
Klotzbucher et al., EMBO J. 1:3053 (1996)).
In a preferred embodiment, the targeting sequence is a secretory signal
sequence capable of effecting the secretion of the candidate protein. There
are a
large number of known secretory signal sequences which are placed 5' to the
variable peptide region, and are cleaved from the peptide region to effect
secretion
into the extracellular space. Secretory signal sequences and their
transferability to
unrelated proteins are well known, e.g., Silhavy, et al. ( 1985) Microbiol.
Rev. 49,
398-418. This is particularly useful to generate a peptide capable of binding
to the
surface of, or affecting the physiology of, a target cell that is other than
the host
cell. In this manner, target cells grown in the vicinity of cells caused to
express
the library of peptides, are bathed in secreted peptide. Target cells
exhibiting a
physiological change in response to the presence of a peptide, e.g., by the
peptide
binding to a surface receptor or by being internalized and binding to
intracellular
targets, and the secreting cells are localized by any of a variety of
selection
schemes and the peptide causing the effect determined. Exemplary effects
include
variously that of a designer cytokine (i.e., a stem cell factor capable of
causing
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34
hematopoietic stem cells to divide and maintain their totipotential), a factor
causing cancer cells to undergo spontaneous apoptosis, a factor that binds to
the
cell surface of target cells and labels them specifically, etc.
Similar to the membrane-anchored embodiment, it is possible that the
formation of the NAP conjugate happens after the screening; that is, having
the
fusion protein secreted means that it may not be available for binding to the
nucleic acid. However, this may be done later, with lysis of the cell.
Suitable secretory sequences are known, including, for example, signals
from IL-2 (see, for example, Villinger et al., J. Immunol. 155:3946 ( 1995)),
growth hormone (see, for example, Roskam et al., Nucleic Acids Res. 7:30
( 1979)); preproinsulin (see, for example, Bell et al., Nature 284:26 (
1980)); and
influenza HA protein (see, for example, Sekiwawa et al., PNAS 80:3563)). A
particularly preferred secretory signal sequence is the signal leader sequence
from
the secreted cytokine IL-4.
In a preferred embodiment, the fusion partner is a rescue sequence
(sometimes also referred to herein as "purification tags" or "retrieval
properties").
A rescue sequence is a sequence which may be used to purify or isolate either
the
candidate protein or the NAP conjugate. Thus, for example, peptide rescue
sequences include purification sequences such as the Hisb tag for use with Ni
affinity columns and epitope tags for detection, immunoprecipitation or FACS
(fluoroscence-activated cell sorting). Suitable epitope tags include myc (for
use
with the commercially available 9E10 antibody), the BSP biotinylation target
sequence of the bacterial enzyme BirA, flu tags, lacZ, and GST. Rescue
sequences
can be utilized on the basis of a binding event, an enzymatic event, a
physical
property or a chemical property.
Alternatively, the rescue sequence can comprise a unique oligonucleotide
sequence which serves as a probe target site to allow the quick and easy
isolation
of the construct, via PCR, related techniques, or hybridization.
In a preferred embodiment, the fusion partner is a stability sequence to
confer stability to the candidate protein or the nucleic acid encoding it.
Thus, for
example, peptides can be stabilized by the incorporation of glycines after the
initiation methionine, for protection of the peptide to ubiquitination as per
Varshavsky's N-End Rule, thus conferring long half life in the cytoplasm.
Similarly, two prolines at the C-terminus impart peptides that are largely
resistant
to carboxypeptidase action. The presence of two glycines prior to the prolines
impart both flexibility and prevent structure initiating events in the di-
proline to be
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propagated into the candidate protein structure. Thus, preferred stability
sequences are as follows: MG(X)~GGPP, where X is any amino acid and n is an
integer of at least four.
In addition, linker sequences, as defined above, may be used in any
configuration as needed.
In addition, the fusion partners, including presentation structures, may be
modified, randomized, and/or matured to alter the presentation orientation of
the
randomized expression product. For example, determinants at the base of the
loop
may be modified to slightly modify the internal loop peptide tertiary
structure,
10 which maintaining the randomized amino acid sequence.
Combinations of fusion partners can be used if desired. Thus, for example,
any number of combinations of presentation structures, targeting sequences,
rescue
sequences, and stability sequences may be used, with or without linker
sequences.
Similarly, as discussed herein, the fusion partners may be associated with any
15 component of the expression vectors described herein: they may be directly
fused
with either the NAM enzyme, the candidate protein, or the EAS, described
below,
or be separate from these components and contained within the expression
vector.
In addition to sequences encoding NAM enzymes and candidate proteins,
and the optional fusion partners, the nucleic acids of the invention
preferably
20 comprise an enzyme attachment sequence. By "enzyme attachment sequence" or
"EAS" herein is meant selected nucleic acid sequences that mediate attachment
with NAM enzymes. Such EAS nucleic acid sequences possess the specific
sequence or specific chemical or structural configuration that allows for
attachment of the NAM enzyme and the EAS. The EAS can comprise DNA or
25 RNA sequences in their natural conformation, or hybrids. EASs also can
comprise
modified nucleic acid sequences or synthetic sequences inserted into the
nucleic
acid molecule of the present invention. EASs also can comprise non-natural
bases
or hybrid non-natural and natural (i.e., found in nature) bases.
As will be appreciated by those in the art, the choice of the EAS will
30 depend on the NAM enzyme, as individual NAM enzymes recognize specific
sequences and thus their use is paired. Thus, suitable NAM/EAS pairs are the
sequences recognized by Rep proteins (sometimes referred to herein as "Rep
EASs") and the Rep proteins, the H-1 recognition sequence and H-l, etc. In
addition, EASs can be utilized which mediate improved covalent binding with
the
35 NAM enzyme compared to the wild-type or naturally occurring EAS.
In a preferred embodiment, the EAS is double-stranded. By way of
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36
example, a suitable EAS is a double-stranded nucleic acid sequence containing
specific features for interacting with corresponding NAM enzymes. For example,
Rep68 and Rep78 recognize an EAS contained within an AAV ITR, the sequence
of which is set forth in Example 1. In addition, these Rep proteins have been
S shown to recognize an ITR-like region in human chromosome 19 as well, the
sequence of which is shown in Figure 48.
An EAS also can comprise supercoiled DNA with which a topoisomerase
interacts and forms covalent intermediate complexes. Alternatively, an EAS is
a
restriction enzyme site recognized by an altered restriction enzyme capable of
forming covalent linkages. Finally, an EAS can comprise an RNA sequence
and/or structure with which specific proteins interact and form stable
complexes
(see, for example, Romaniuk and Uhlenbeck, Biochemistry, 24, 4239-44 ( 1985)).
The present invention relies on the specific binding of the NAM enzyme to
the EAS in order to mediate linkage of the fusion enzyme to the nucleic acid
molecule. One of ordinary skill in the art will appreciate that use of an EAS
consisting of a small nucleic acid sequence would result in non-specific
binding of
the NAM enzyme to expression vectors and the host cell genome depending on the
frequency that the accessible EAS motif appears in the vector or host genome.
Therefore, the EAS of the present invention is preferably comprised of a
nucleic
acid sequence of sufficient length such that specific fusion protein-coding
nucleic
acid molecule attachment results. For example, the EAS is preferably greater
than
five nucleotides in length. More preferably, the EAS is greater than 10
nucleotides
in length, e.g., with EASs of at least 12, 15, 20, 25, 30, 35, 40, 45 or 50
nucleotides being preferred.
Moreover, preferably the EAS is present in the host cell genome in a very
limited manner, such that at most, only one or two NAM enzymes can bind per
genome, e.g. no more than once in a human cell genome. In situations wherein
the
EAS is present many times within a host cell, e.g., a human cell genome, the
probability of fusion proteins encoded by the expression vector attaching to
the
host cell genome and not the expression vector increases and is therefore
undesirable. For instance, the bacteriophage P2 A protein recognizes a
relatively
short DNA recognition sequence. As such, use of the P2 A protein in mammalian
cells would result in protein binding throughout the host genome, and
identification of the desired nucleic acid sequence would be difficult. Thus,
preferred embodiments exclude the use of P2A as a NAM enzyme.
One of ordinary skill in the art will appreciate that the NAM enzyme used
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37
in the present invention or the corresponding EAS can be manipulated in order
to
increase the stability of the fusion protein-nucleic acid molecule complex.
Such
manipulations are contemplated herein, so long as the NAM enzyme forms a
covalent bond with its corresponding EAS.
S Thus, in a preferred embodiment, the nucleic acids of the invention
comprise (i) a fusion nucleic acid comprising sequences encoding a NAM enzyme
and a candidate protein, and (ii) an EAS. These nucleic acids are preferably
incorporated into an expression vector; thus providing libraries of expression
vectors, sometimes referred to herein as "NAM enzyme expression vectors".
The expression vectors may be either self replicating extrachromosomal
vectors, vectors which integrate into a host genome, or linear nucleic acids
that
may or may not self replicate. Thus, specifically included within the
definition of
expression vectors are linear nucleic acid molecules. Expression vectors thus
include plasmids, plasmid-liposome complexes, phage vectors, and viral
vectors,
e.g., adeno-associated virus (AAV)-based vectors, retroviral vectors, herpes
simplex virus (HSV)-based vectors, and adenovirus-based vectors. The nucleic
acid molecule and any of these expression vectors can be prepared using
standard
recombinant DNA techniques described in, for example, Sambrook et al.,
Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press,
Cold Spring Harbor, N.Y. ( 1989), and Ausubel et al., Current Protocols in
Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New
York, N.Y. (1994) Generally, these expression vectors include transcriptional
and
translational regulatory nucleic acid sequences operably linked to the nucleic
acid
encoding the NAM protein. The term "control sequences" refers to DNA
sequences necessary for the expression of an operably linked coding sequence
in a
particular host organism. The control sequences that are suitable for
prokaryotes,
for example, include a promoter, optionally an operator sequence, and a
ribosome
binding site. Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
A nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic acid sequence. For example, DNA for a
presequence or secretory leader is operably linked to DNA encoding a
polypeptide
if it is expressed as a preprotein that participates in the secretion of the
polypeptide; a promoter or enhancer is operably linked to a coding sequence if
it
affects the transcription of the sequence; or a ribosome binding site is
operably
linked to a coding sequence if it is positioned so as to facilitate
translation.
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38
Generally, "operably linked" means that the DNA sequences being linked are
contiguous, and, in the case of a secretory leader, contiguous and in reading
phase.
However, enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not exist, the
synthetic
oligonucleotide adaptors or linkers are used in accordance with conventional
practice. The transcriptional and translational regulatory nucleic acid will
generally be appropriate to the host cell used to express the NAM protein, as
will
be appreciated by those in the art; for example, transcriptional and
translational
regulatory nucleic acid sequences from Bacillus are preferably used to express
the
NAM protein in Bacillus. Numerous types of appropriate expression vectors, and
suitable regulatory sequences are known in the art for a variety of host
cells.
In general, the transcriptional and translational regulatory sequences may
include, but are not limited to, promoter sequences, ribosomal binding sites,
transcriptional start and stop sequences, translational start and stop
sequences, and
enhancer, silencer, or activator sequences. In a preferred embodiment, the
regulatory sequences include a promoter and transcriptional start and stop
sequences.
A "promoter" is a nucleic acid sequence that directs the binding of RNA
polymerase and thereby promotes RNA synthesis. Promoter sequences include
constitutive and inducible promoter sequences. Exemplary constitutive
promoters
include, but are not limited to, the CMV immediate-early promoter, the RSV
long
terminal repeat, mouse mammary tumor virus (MMTV) promoters, etc. Suitable
inducible promoters include, but are not limited to, the IL-8 promoter, the
metallothionine inducible promoter system, the bacterial IacZYA expression
system, the tetracycline expression system, and the T7 polyrnerases system.
The
promoters can be either naturally occurring promoters, hybrid promoters, or
synthetic promoters. Hybrid promoters, which combine elements of more than one
promoter, are also known in the art, and are useful in the present invention.
In addition, the expression vector may comprise additional elements. For
example, the expression vector may have two replication systems (e.g., origins
of
replication), thus allowing it to be maintained in two organisms, for example
in
animal cells for expression and in a prokaryotic host for cloning and
amplification.
Furthermore, for integrating expression vectors, which are generally not
preferred
in most embodiments, the expression vector contains at least one sequence
homologous to the host cell genome, and preferably two homologous sequences
which flank the expression construct. The integrating vector may be directed
to a
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39
specific locus in the host cell by selecting the appropriate homologous
sequence
for inclusion in the vector. Constructs for integrating vectors and
appropriate
selection and screening protocols are well known in the art and are described
in
e.g., Mansour et al., Cell, 51:503 (1988) and Murray, Gene Transfer and
S Expression Protocols, Methods in Molecular Biology, Vol. 7 (Clifton: Humana
Press, 1991 ).
It should be noted that the compositions and methods of the present
invention allow for specific chromosomal isolation. For example, since human
chromosome 19 contains a Rep-binding sequence (e.g. an EAS), a NAP conjugate
will be formed with chromosome 19, when the NAM enzyme is Rep. Cell lysis
followed by immunoprecipitation, either using antibodies to the Rep protein
itself
(e.g. no candidate protein is necessary) or to a fused candidate protein or
purification tag, allows the purification of the chromosome. This is a
significant
advance over current chromosome purification techniques. Thus, by selectively
or
non-selectively integrating EAS sites into chromosomes, different chromosomes
may be purified.
In addition, in a preferred embodiment, the expression vector contains a
selection gene to allow the selection of transformed host cells containing the
expression vector, and particularly in the case of mammalian cells, ensures
the
stability of the vector, since cells which do not contain the vector will
generally
die. Selection genes are well known in the art and will vary with the host
cell
used. By "selection gene" herein is meant any gene which encodes a gene
product
that confers new phenotypes of the cells which contain the vector. These
phenotypes include, for instance, enhanced or decreased cell growth. The
phenotypes can also include resistance to a selection agent. Suitable
selection
agents include, but are not limited to, neomycin (or its analog G418),
blasticidin S,
histinidol D, bleomycin, puromycin, hygromycin B, and other drugs. The
expression vector also can comprise a coding sequence for a marker protein,
such
as the green fluorescence protein, which enables, for example, rapid
identification
of successfully transduced cells.
In a preferred embodiment, the expression vector contains a RNA splicing
sequence upstream or downstream of the gene to be expressed in order to
increase
the level of gene expression. See Barret et al., Nucleic Acids Res. 1991;
Groos et
al., Mol. Cell. Biol. 1987; and Budiman et al., Mol. Cell. Biol. 1988.
One expression vector system is a retroviral vector system such as is
generally described in Mann et al., Cell, 33:153-9 (1993); Pear et al., Proc.
Natl.
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Acad. Sci. U.S.A., 90(18):8392-6 (1993); Kitamura et al., Proc. Natl. Acad.
Sci.
U.S.A., 92:9146-50 (1995); Kinsella et al., Human Gene Therapy, 7:1405-13;
Hofmann et al.,Proc. Natl. Acad. Sci. U.S.A., 93:5185-90; Choate et al., Human
Gene Therapy, 7:2247 ( 1996); PCT/LJS97/01019 and PCT/LTS97/01048, and
references cited therein, all of which are hereby expressly incorporated by
reference.
The fusion proteins of the present invention can be produced by culturing a
host cell transformed with nucleic acid, preferably an expression vector as
outlined
herein, under the appropriate conditions to induce or cause production of the
10 fusion protein. The conditions appropriate for fusion protein production
will vary
with the choice of the expression vector and the host cell, and will be easily
ascertained by one skilled in the art using routine methods. For example, the
use
of constitutive promoters in the expression vector will require optimizing the
growth and proliferation of the host cell, while the use of an inducible
promoter
15 requires the appropriate growth conditions for induction. In addition, in
some
embodiments, the timing of the harvest is important. For example, the
baculoviral
systems used in insect cells are lytic viruses, and thus harvest time
selection can be
crucial for product yield.
Any host cell capable of withstanding introduction of exogenous DNA and
20 subsequent protein production is suitable for the present invention. The
choice of
the host cell will depend, in part, on the assay to be run; e.g., in vitro
systems may
allow the use of any number of procaryotic or eucaryotic organisms, while ex
vivo
systems preferably utilize animal cells, particularly mammalian cells with a
special
emphasis on human cells. Thus, appropriate host cells include yeast, bacteria,
25 archaebacteria, plant, and insect and animal cells, including mammalian
cells and
particularly human cells. The host cells may be native cells, primary cells,
including those isolated from diseased tissues or organisms, cell lines (again
those
originating with diseased tissues), genetically altered cells, etc. Of
particular
interest are Drosophila melanogaster cells, Saccharomyces cerevisiae and other
30 yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells,
Neurospora,
BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanoma cell lines, etc. See the
ATCC cell line catalog, hereby expressly incorporated by reference.
In a preferred embodiment, the fusion proteins are expressed in mammalian
cells. Mammalian expression systems are also known in the art, and include,
for
35 example, retroviral and adenoviral systems. A mammalian promoter is any DNA
sequence capable of binding mammalian RNA polymerase and initiating the
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41
downstream (3') transcription of a coding sequence for a fusion protein into
mRNA. A promoter will have a transcription initiating region, which is usually
placed proximal to the 5' end of the coding sequence, and a TATA box, using a
located 25-30 base pairs upstream of the transcription initiation site. The
TATA
box is thought to direct RNA polymerase II to begin RNA synthesis at the
correct
site. A mammalian promoter will also contain an upstream promoter element
(enhancer element), typically located within 100 to 200 base pairs upstream of
the
TATA box. An upstream promoter element determines the rate at which
transcription is initiated and can act in either orientation. Of particular
use as
mammalian promoters are the promoters from mammalian viral genes, since the
viral genes are often highly expressed and have a broad host range. Examples
include the SV40 early promoter, mouse mammary tumor virus LTR promoter,
adenovirus major late promoter, herpes simplex virus promoter, and the CMV
promoter.
Typically, transcription termination and polyadenylation sequences
recognized by mammalian cells are regulatory regions located 3' to the
translation
stop codon and thus, together with the promoter elements, flank the coding
sequence. The 3' terminus of the mature mRNA is formed by site-specific post-
translational cleavage and polyadenylation. Examples of transcription
terminator
and polyadenlytion signals include those derived from SV40.
The methods of introducing exogenous nucleic acid into mammalian hosts,
as well as other hosts, is well known in the art, and will vary with the host
cell
used. Techniques include dextran-mediated transfection, calcium phosphate
precipitation, polybrene mediated transfection, protoplast fusion,
electroporation,
viral infection, encapsulation of the polynucleotide(s) in liposomes, and
direct
microinjection of the DNA into nuclei.
In a preferred embodiment, NAM fusions are produced in bacterial
systems. Bacterial expression systems are widely available and include, for
example, plasmids.
A suitable bacterial promoter is any nucleic acid sequence capable of
binding bacterial RNA polymerase and initiating the downstream (3')
transcription
of the coding sequence of the fusion into mRNA. A bacterial promoter has a
transcription initiation region which is usually placed proximal to the 5' end
of the
coding sequence. This transcription initiation region typically includes an
RNA
polymerase binding site and a transcription initiation site. Sequences
encoding
metabolic pathway enzymes provide particularly useful promoter sequences.
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42
Examples include promoter sequences derived from sugar metabolizing enzymes,
such as galactose, lactose and maltose, and sequences derived from
biosynthetic
enzymes such as tryptophan. Promoters from bacteriophage may also be used and
are known in the art. In addition, synthetic promoters and hybrid promoters
are
also useful; for example, the tac promoter is a hybrid of the trp and lac
promoter
sequences. Furthermore, a bacterial promoter can include naturally occurring
promoters of non-bacterial origin that have the ability to bind bacterial RNA
polymerase and initiate transcription.
In addition to a functioning promoter sequence, an efficient ribosome
binding site is desirable. In E. coli, the ribosome binding site is called the
Shine-
Delgarno (SD) sequence and includes an initiation codon and a sequence 3-9
nucleotides in length located 3 - 11 nucleotides upstream of the initiation
codon.
The expression vector may also include a signal peptide sequence that
provides for secretion of the fusion proteins in bacteria or other cells. The
signal
sequence typically encodes a signal peptide comprised of hydrophobic amino
acids
which direct the secretion of the protein from the cell, as is well known in
the art.
The protein is either secreted into the growth media (gram-positive bacteria)
or
into the periplasmic space, located between the inner and outer membrane of
the
cell (gram-negative bacteria).
The bacterial expression vector may also include a selectable marker gene
to allow for the selection of bacterial strains that have been transformed.
Suitable
selection genes include genes which render the bacteria resistant to drugs
such as
ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and
tetracycline. Selectable markers also include biosynthetic genes, such as
those in
the histidine, tryptophan and leucine biosynthetic pathways.
Suitable bacterial cells include, for example, vectors for Bacillus subtilis,
E.
coli, Streptococcus cremoris, and Streptococcus lividans, among others. The
bacterial expression vectors can be transformed into bacterial host cells
using
techniques well known in the art, such as calcium chloride treatment,
electroporation, and others. One benefit of using bacterial cells in the
ability to
propagate the cells comprising the expression vectors, thus generating clonal
populations.
NAM fusion proteins also can be produced in insect cells such as S~ cells.
Expression vectors for the transformation of insect cells, and in particular,
baculovirus-based expression vectors, are well known in the art and are
described
e.g., in O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual
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43
(New York: Oxford University Press, 1994).
In addition, NAM fusion proteins can be produced in yeast cells. Yeast
expression systems are well known in the art, and include, for example,
expression
vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa,
Hansenula polymorpha, Kluyveromyces fragilis and K lactis, Pichia
guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia
lipolytica. Preferred promoter sequences for expression in yeast include the
inducible GAL 1,10 promoter, the promoters from alcohol dehydrogenase,
enolase,
glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-
dehydrogenase, hexokinase, phosphofructokinase, 3-phosphoglycerate mutase,
pyruvate kinase, and the acid phosphatase gene. Yeast selectable markers
include
ADE2, HIS4, LEU2, TRP 1, and ALG7, which confers resistance to tunicamycin;
the neomycin phosphotransferase gene, which confers resistance to 6418; and
the
CUP1 gene, which allows yeast to grow in the presence of copper ions. One
benefit of using yeast cells is the ability to propagate the cells comprising
the
vectors, thus generating clonal populations.
Preferred expression vectors are shown in Figures 49A-49N.
In addition to the components outlined herein, including NAM enzyme-
candidate protein fusions, EASs, linkers, fusion partners, etc., the
expression
vectors may comprise a number of additional components, including, selection
genes as outlined herein (particularly including growth-promoting or growth-
inhibiting functions), activatible elements, recombination signals (e.g. cre
and lox
sites) and labels.
Preferably, the present invention fusion peptide, fusion nucleic acid,
conjugates, etc., further comprise a labeling component. Again, as for the
fusion
partners of the invention, the label can be fused to one or more of the other
components, for example to the NAM fusion protein, in the case where the NAM
enzyme and the candidate protein remain attached, or to either component, in
the
case where scission occurs, or separately, under its own promoter. In
addition, as
is further described below, other components of the assay systems may be
labeled.
Labels can be either direct or indirect detection labels, sometimes referred
to herein as "primary" and "secondary" labels. By "detection label" or
"detectable
label" herein is meant a moiety that allows detection. This may be a primary
label
or a secondary label. Accordingly, detection labels may be primary labels
(i.e.
directly detectable) or secondary labels (indirectly detectable).
In general, labels fall into four classes: a) isotopic labels, which may be
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44
radioactive or heavy isotopes; b) magnetic, electrical, thermal labels; c)
colored or
luminescent dyes or moieties; and d) binding partners. Labels can also include
enzymes (horseradish peroxidase, etc.) and magnetic particles. In a preferred
embodiment, the detection label is a primary label. A primary label is one
that can
be directly detected, such as a fluorophore.
Preferred labels include, for example, chromophores or phosphors but are
preferably fluorescent dyes or moieties. Fluorophores can be either "small
molecule" fluors, or proteinaceous fluors. In a preferred embodiment,
particularly
for labeling of target molecules, as described below, suitable dyes for use in
the
invention include, but are not limited to, fluorescent lanthanide complexes,
including those of Europium and Terbium, fluorescein, rhodamine,
tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, quantum
dots (also referred to as "nanocrystals"), pyrene, Malacite green, stilbene,
Lucifer
Yellow, Cascade BlueT"'', Texas Red, Cy dyes (Cy3, CyS, etc.), alexa dyes,
1 S phycoerythin, bodipy, and others described in the 6th Edition of the
Molecular
Probes Handbook by Richard P. Haugland, hereby expressly incorporated by
reference.
In a preferred embodiment, for example when the label is attached to the
fusion polypeptide or is to be expressed as a component of the expression
vector,
proteinaceous fluores are used. Suitable autofluorescent proteins include, but
are
not limited to, the green fluorescent protein (GFP) from Aequorea and variants
thereof; including, but not limited to, GFP, (Chalfie, et al., Science
263(5148):802-
805 ( 1994)); enhanced GFP (EGFP; Clontech - Genbank Accession Number
U55762 )), blue fluorescent protein (BFP; Quantum Biotechnologies, Inc. 1801
de
Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1J9;
Stauber, R. H. Biotechniques 24(3):462-471 (1998); Heim, R. and Tsien, R. Y.
Curr. Biol. 6:178-182 (1996)), and enhanced yellow fluorescent protein (EYFP;
Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA 94303). In
addition, there are recent reports of autofluorescent proteins from Renilla
species.
See WO 92/15673; WO 95/07463; WO 98/14605; WO 98/26277; WO 99/49019;
U.S. patent 5,292,658; U.S patent 5,418,155; U.S. patent 5,683,888; U.S.
patent
5,741,668; U.S. patent 5,777,079; U.S. patent 5,804,387; U.S. patent
5,874,304;
U.S patent 5,876,995; and U.S. patent 5,925,558; all of which are expressly
incorporated herein by reference.
In a preferred embodiment, the label protein is Aequorea green fluorescent
protein or one of its variants; see Cody et al., Biochemistry 32:1212-1218
(1993);
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and Inouye and Tsuji, FEBS Lett. 341:277-280 (1994), both of which are
expressly incorporated by reference herein.
In a preferred embodiment, a secondary detectable label is used. A
secondary label is one that is indirectly detected; for example, a secondary
label
5 can bind or react with a primary label for detection, can act on an
additional
product to generate a primary label (e.g. enzymes), or may allow the
separation of
the compound comprising the secondary label from unlabeled materials, etc.
Secondary labels include, but are not limited to, one of a binding partner
pair;
chemically modifiable moieties; enzymes such as horseradish peroxidase,
alkaline
10 phosphatases, luciferases, etc; and cell surface markers, etc.
In a preferred embodiment, the secondary label is a binding partner pair.
For example, the label may be a hapten or antigen, which will bind its binding
partner. In a preferred embodiment, the binding partner can be attached to a
solid
support to allow separation of components containing the label and those that
do
15 not. For example, suitable binding partner pairs include, but are not
limited to:
antigens (such as proteins (including peptides)) and antibodies (including
fragments thereof (FAbs, etc.)); proteins and small molecules, including
biotin/streptavidin; enzymes and substrates or inhibitors; other protein-
protein
interacting pairs; receptor-ligands; and carbohydrates and their binding
partners.
20 Nucleic acid - nucleic acid binding proteins pairs are also useful. In
general, the
smaller of the pair is attached to the system component for incorporation into
the
assay, although this is not required in all embodiments. Preferred binding
partner
pairs include, but are not limited to, biotin (or imino-biotin) and
streptavidin,
digeoxinin and Abs, etc.
25 In a preferred embodiment, the binding partner pair comprises a primary
detection label (for example, attached to the assay component) and an antibody
that will specifically bind to the primary detection label. By "specifically
bind"
herein is meant that the partners bind with specificity sufficient to
differentiate
between the pair and other components or contaminants of the system. The
30 binding should be sufficient to remain bound under the conditions of the
assay,
including wash steps to remove non-specific binding. In some embodiments, the
dissociation constants of the pair will be less than about 10~-10-6 M~~, with
less
than about 10-5-10-9 M-', being preferred and less than about 10-'-10-9 M-~
being
particularly preferred.
35 In a preferred embodiment, the secondary label is a chemically modifiable
moiety. In this embodiment, labels comprising reactive functional groups are
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46
incorporated into the assay component. The functional group can then be
subsequently labeled with a primary label. Suitable functional groups include,
but
are not limited to, amino groups, carboxy groups, maleimide groups, oxo groups
and thiol groups, with amino groups and thiol groups being particularly
preferred.
For example, primary labels containing amino groups can be attached to
secondary
labels comprising amino groups, for example using linkers as are known in the
art;
for example, homo-or hetero-bifunctional linkers as are well known (see 1994
Pierce Chemical Company catalog, technical section on cross-linkers, pages
155-200, incorporated herein by reference).
It can be advantageous to construct the expression vector to provide further
options to control attachment of the fusion enzyme to the EAS. For example,
the
EAS can be introduced into the nucleic acid molecule as two non-functional
halves
that are brought together following enzyme-mediated or non-enzyme-mediated
homologous recombination, such as that mediated by cre-lox recombination, to
1 S form a functional EAS. Likewise, the referenced cre-lox consideration
could also
be used to control the formation of a functional fusion enzyme. The control of
cre-
lox recombination is preferably mediated by introducing the recombinase gene
under the control of an inducible promoter into the expression system, whether
on
the same nucleic acid molecule or on another expression vector.
In general, once the expression vectors of the invention are made, they can
follow one of two fates, which are merely exemplary: they are introduced into
cell-
free translation systems, to create libraries of nucleic acid/protein (NAP)
conjugates that are assayed in vitro, or, preferably they are introduced into
host
cells where the NAP conjugates are formed; the cells may be optionally lysed
and
assayed accordingly.
In a preferred embodiment, the expression vectors are made and introduced
into cell-free systems for translation, followed by the attachment of the NAP
enzyme to the EAS, forming a nucleic acid/protein (NAP) conjugate. By "nucleic
acid/protein conjugate" or "NAP conjugate" herein is meant a covalent
attachment
between the NAP enzyme and the EAS, such that the expression vector comprising
the EAS is covalently attached to the NAP enzyme. Suitable cell free
translation
systems are known in the art. Once made, the NAP conjugates are used in assays
as outlined below.
In a preferred embodiment, the expression vectors of the invention are
introduced into host cells as outlined herein. By "introduced into" or
grammatical
equivalents herein is meant that the nucleic acids enter the cells in a manner
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47
suitable for subsequent expression of the nucleic acid. The method of
introduction
is largely dictated by the targeted cell type, discussed below. Exemplary
methods
include CaP04 precipitation, liposome fusion, lipofectin~, electroporation,
viral
infection, gene guns, etc. The candidate nucleic acids may stably integrate
into the
genome of the host cell (for example, with retroviral introduction, outlined
herein)
or may exist either transiently or stably in the cytoplasm (i.e. through the
use of
traditional plasmids, utilizing standard regulatory sequences, selection
markers,
etc.). Suitable host cells are outlined above, with eucaryotic, mammalian and
human cells all preferred.
Many previously described methods involve peptide library expression in
bacterial cells. Yet, it is understood in the art that translational machinery
such as
codon preference, protein folding machinery, and post-translational
modifications
of, for example, mammalian peptides, are unachievable or altered in bacterial
cells,
if such modifications occur at all. Peptide library screening in bacterial
cells often
1 S involves expression of short amino acid sequences, which can not imitate a
protein
in its natural configuration. Screening of these small, sub-part sequences
cannot
effectively determine the function of a native protein in that the
requirements for,
for instance, recognition of a small ligand for its receptor, are easily
satisfied by
small sequences without native conformation. The complexities of tertiary
structure are not accounted for, thereby easing the requirements for binding.
One advantage of the present invention is the ability to express and screen
unknown peptides in their native environment and in their native protein
conformation. The covalent attachment of the fusion enzyme to its
corresponding
expression vector allows screening of peptides in organisms other than
bacteria.
Once introduced into a eukaryotic host cell, the nucleic acid molecule is
transported into the nucleus where replication and transcription occurs. The
transcription product is transferred to the cytoplasm for translation and post-
translational modifications. However, the produced peptide and corresponding
nucleic acid molecule must meet in order for attachment to occur, which is
hindered by the compartmentalization of eukaryotic cells. NAM enzyme-EAS
recognition can occur in four ways, which are merely exemplary and do not
limit
the present invention in any way. First, the host cells can be allowed to
undergo
one round of division, during which the nuclear envelope breaks down. Second,
the host cells can be infected with viruses that perforate the nuclear
envelope.
Third, specific nuclear localization or transporting signals can be introduced
into
the fusion enzyme. Finally, host cell organelles can be disrupted using
methods
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48
known in the art.
The end result of the above-described approaches is the transfer of the
expression vector into the same environment as the fusion enzyme. The non
covalent interaction between a DNA binding protein and attachment site of
previously described expression libraries would not survive the procedures
required to allow linkage of the fusion protein to its expression vector in
eukaryotic cells. Other DNA-protein linkages described in the art, such as
those
using the bacterial P2 A DNA binding peptide, require the binding peptide to
remain in direct contact with its coding DNA in order for binding to occur,
i.e.,
translation must occur proximal to the coding sequence (see, for example,
Lindahl,
Virology, 42, 522-533 ( 1970)). Such linkages are only achievable in
prokaryotic
systems and cannot be produced in eukaryotic cells.
Once the NAM enzyme expression vectors have been introduced into the
host cells, the cells are optionally lysed. Cell lysis is accomplished by any
suitable
technique, such as any of a variety of techniques known in the art (see, for
example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. ( 1989), and Ausubel et
al.,
Current Protocols in Molecular Biology, Greene Publishing Associates and John
Wiley & Sons, New York, N.Y. ( 1994), hereby expressly incorporated by
reference). Most methods of cell lysis involve exposure to chemical,
enzymatic, or
mechanical stress. Although the attachment of the fusion enzyme to its coding
nucleic acid molecule is a covalent linkage, and can therefore withstand more
varied conditions than non-covalent bonds, care should be taken to ensure that
the
fusion enzyme-nucleic acid molecule complexes remain intact, i.e., the fusion
enzyme remains associated with the expression vector.
In a preferred embodiment, the NAP conjugate may be purified or isolated
after lysis of the cells. Ideally, the lysate containing the fusion protein-
nucleic acid
molecule complexes is separated from a majority of the resulting cellular
debris in
order to facilitate interaction with the target. For example, the NAP
conjugate
may be isolated or purified away from some or all of the proteins and
compounds
with which it is normally found after expression, and thus may be
substantially
pure. For example, an isolated NAP conjugate is unaccompanied by at least some
of the material with which it is normally associated in its natural
(unpurified) state,
preferably constituting at least about 0.5%, more preferably at least about S%
by
weight or more of the total protein in a given sample. A substantially pure
protein
comprises at least about 75% by weight or more of the total protein, with at
least
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49
about 80% or more being preferred, and at least about 90% or more being
particularly preferred.
NAP conjugates may be isolated or purified in a variety of ways known to
those skilled in the art depending on what other components are present in the
S sample. Standard purification methods include electrophoretic, molecular,
immunological and chromatographic techniques, including ion exchange,
hydrophobic, affinity, and reverse-phase HPLC chromatography, gel filtration,
and
chromatofocusing. Ultrafiltration and diafiltration techniques, in conjunction
with
protein concentration, are also useful. For general guidance in suitable
purification techniques, see Scopes, R., Protein Purification, Springer-
Verlag, NY
( 1982). The degree of purification necessary will vary depending on the use
of the
NAP conjugate. In some instances no purification will be necessary.
Thus, the invention provides for NAP conjugates that are either in solution,
optionally purified or isolated, or contained within host cells. Once
expressed and
purified if necessary, the NAP conjugates are useful in a number of
applications,
including in vitro and ex vivo screening techniques. One of ordinary skill in
the
art will appreciate that both in vitro and ex vivo embodiments of the present
inventive method have utility in a number of fields of study. For example, the
present invention has utility in diagnostic assays and can be employed for
research
in numerous disciplines, including, but not limited to, clinical pharmacology,
functional genomics, pharamcogenomics, agricultural chemicals, environmental
safety assessment, chemical sensor, nutrient biology, cosmetic research, and
enzymology.
In a preferred embodiment, the NAP conjugates are used in in vitro
screening techniques. In this embodiment, the NAP conjugates are made and
screened for binding and/or modulation of bioactivites of target molecules.
One of
the strengths of the present invention is to allow the identification of
target
molecules that bind to the candidate proteins. As is more fully outlined
below, this
has a wide variety of applications, including elucidating members of a
signaling
pathway, elucidating the binding partners of a drug or other compound of
interest,
etc.
Thus, the NAP conjugates are used in assays with target molecules. By
"target molecules" or grammatical equivalents herein is meant a molecule for
which an interaction is sought; this term will be generally understood by
those in
the art. Target molecules include both biological and non-biological targets.
Biological targets refer to any defined and non-defined biological particles,
such as
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macromolecular complexes, including viruses, cells, tissues and combinations,
that
are produced as a result of biological reactions in cells. Non-biological
targets
refer to molecules or structure that are made outside of cells as a result of
either
human or non-human activity. The inventive library can also be applied to both
chemically defined targets and chemically non-defined targets. "Chemically
defined targets" refer to those targets with known chemical nature and/or
composition; "chemically non-defined targets" refer to targets that have
either
unknown or partially known chemical nature/composition.
Thus, suitable target molecules encompass a wide variety of different
classes, including, but not limited to, cells, viruses, proteins (particularly
including
enzymes, cell-surface receptors, ion channels, and transcription factors, and
proteins produced by disease-causing genes or expressed during disease
states),
carbohydrates, fatty acids and lipids, nucleic acids, chemical moieties such
as
small molecules, agricultural chemicals, drugs, ions (particularly metal
ions),
polymers and other biomaterials. Thus for example, binding to polymers (both
naturally occurring and synthetic), or other biomaterials, may be done using
the
methods and compositions of the invention.
In one aspect, the target is a nucleic acid sequence and the desired
candidate protein has the ability to bind to the nucleic acid sequence. The
present
invention is well suited for identification of DNA binding peptides and their
coding sequences, as well as the target nucleic acids that are recognized and
bound
by the DNA binding peptides. It is known that DNA-protein interactions play
important roles in controlling gene expression and chromosomal structure,
thereby
determining the overall genetic program in a given cell. It is estimated that
only
5% of the human genome is involved in coding proteins. Thus, the remaining 95%
may be sites with which DNA binding proteins interact, thereby controlling a
variety of genetic programs such as regulation of gene expression. While the
number of DNA binding peptides present in the human genome is not known, the
complete sequence information now available for many genomes has revealed the
full "substrate," that is, the entire repertoire of DNA sequences with which
DNA
binding peptides may interact. Thus, it would be advantageous in genetic
research
to ( 1 ) identify nucleic acid sequences that encode DNA binding peptides, and
(2)
determine the substrate of these DNA binding peptides.
Current approaches used in determining protein-DNA interactions are
focused on studying the individual interactions between DNA and specific
protein
targets. A variety of biochemical and molecular assays including DNA
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footprinting, nuclease protection, gel shift, and affinity chromatographic
binding
are employed to study protein-DNA interactions. Although these methods are
useful for detecting individual DNA-protein interactions, they are not
suitable for
large-scale analyses of these interactions at the genomic level. Thus, there
is a
S need in the art to perform large-scale analyses of DNA binding proteins and
their
interacting DNA sequences. The methods and libraries of the present invention
are useful for such analyses. For example, the fusion enzyme library encoding
potential DNA binding peptides can be screened against a population of target
DNA segments. The population of target DNA segments can be, for instance,
random DNA, fragmented genomic DNA, degenerate sequences, or DNA
sequences of various primary, secondary or tertiary structures. The
specificity of
the DNA binding peptide-substrate binding can be varied by changing the length
of the recognition sequence of the target DNA, if desired. Binding of the
potential
DNA binding peptide to a member of the population of target DNA segments is
1 S detected, and further study of the particular DNA recognition sequence
bound by
the DNA binding peptide can be performed. To facilitate identification of
fusion
enzyme-target nucleic acid complexes, the population of DNA segments can be
bound to, for example, beads or constructed as DNA arrays on microchips.
Therefore, using the present inventive method, one of ordinary skill in the
art can
identify DNA binding peptides, identify the coding sequence of the DNA binding
peptides, and determine what nucleic acid sequence the DNA binding peptides
recognize and bind. Thus, in one embodiment, the present invention provides
methods for creating a map of DNA binding sequences and DNA binding proteins
according to their relative positions, to provide chromosome maps annotated
with
proteins and sequences. A database comprising such information would then
allow for correlating gene expression profiles, disease phenotype,
pharmacogenomic data, and the like.
Thus, the NAP conjugates are used in screens to assay binding to target
molecules and/or to screen candidate agents for the ability to modulate the
activity
of the target molecule.
In general, screens are designed to first find candidate proteins that can
bind to target molecules, and then these proteins are used in assays that
evaluate
the ability of the candidate protein to modulate the target's bioactivity.
Thus,
there are a number of different assays which may be run; binding assays and
activity assays. As will be appreciated by those in the art, these assays may
be run
in a variety of configurations, including both solution-based assays and
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utilizing support-based systems.
In a preferred embodiment, the assays comprise combining the NAP
conjugates of the invention and a target molecule, and determining the binding
of
the candidate protein of the NAP conjugate to the target molecule. Preferably,
libraries of NAP conjugates (e.g. comprising a library of different candidate
proteins) is contacted with either a single type of target molecule, a
plurality of
target molecules, or one or more libraries of target molecules.
Generally, in a preferred embodiment of the methods herein, one of the
components of the invention, either the NAP conjugate or the target molecule,
is
non-diffusably bound to an insoluble support having isolated sample receiving
areas (e.g. a microtiter plate, an array, etc.). The insoluble support may be
made
of any composition to which the assay component can be bound, is readily
separated from soluble material, and is otherwise compatible with the overall
method of screening. The surface of such supports may be solid or porous and
of
any convenient shape. Examples of suitable insoluble supports include
microtiter
plates, arrays, membranes and beads. These are typically made of glass,
plastic
(e.g., polystyrene), polysaccharides, nylon or nitrocellulose, teflonTM, etc.
Microtiter plates and arrays are especially convenient because a large number
of
assays can be carried out simultaneously, using small amounts of reagents and
samples. Alternatively, bead-based assays may be used, particularly with use
with
fluorescence activated cell sorting (FACS). The particular manner of binding
the
assay component is not crucial so long as it is compatible with the reagents
and
overall methods of the invention, maintains the activity of the composition
and is
nondiffusable. Preferred methods of binding include the use of antibodies
(which
do not sterically block either the ligand binding site or activation sequence
when
the protein is bound to the support), direct binding to "sticky" or ionic
supports,
chemical crosslinking, the use of labeled components (e.g. the assay component
is
biotinylated and the surface comprises strepavidin, etc.) the synthesis of the
target
on the surface, etc. Following binding of the NAP conjugate or target
molecule,
excess unbound material is removed by suitable methods including, for example,
chemical, physical, and biological separation techniques. The sample receiving
areas may then be blocked through incubation with bovine serum albumin (BSA),
casein or other innocuous protein or other moiety.
In a preferred embodiment, the target molecule is bound to the support, and
a NAP conjugate is added to the assay. Alternatively, the NAP conjugate is
bound
to the support and the target molecule is added. Novel binding agents include
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specific antibodies, non-natural binding agents identified in screens of
chemical
libraries, peptide analogs, etc. Of particular interest are screening assays
for
agents that have a low toxicity for human cells. Determination of the binding
of
the target and the candidate protein is done using a wide variety of assays,
S including, but not limited to labeled in vitro protein-protein binding
assays,
electrophoretic mobility shift assays, immunoassays for protein binding, the
detection of labels, functional assays (phosphorylation assays, etc.) and the
like.
The determination of the binding of the candidate protein to the target
molecule may be done in a number of ways. In a preferred embodiment, one of
the
components, preferably the soluble one, is labeled, and binding determined
directly by detection of the label. For example, this may be done by attaching
the
NAP conjugate to a solid support, adding a labeled target molecule (for
example a
target molecule comprising a fluorescent label), removing excess reagent, and
determining whether the label is present on the solid support. This system may
also be run in reverse, with the target (or a library of targets) being bound
to the
support and a NAP conjugate, preferably comprising a primary or secondary
label,
is added. For example, NAP conjugates comprising fusions with GFP or a variant
may be particularly useful. Various blocking and washing steps may be utilized
as
is known in the art.
As will be appreciated by those in the art, it is also possible to contact the
NAP conjugates and the targets prior to immobilization on a support.
In a preferred embodiment, the solid support is in an array format; that is, a
biochip is used which comprises one or more libraries of either targets or NAP
conjugates attached to the array. This can find particular use in assays for
nucleic
acid binding proteins, as nucleic acid biochips are well known in the art. In
this
embodiment, the nucleic acid targets are on the array and the NAP conjugates
are
added. Similarly, protein biochips of libraries of target proteins can be
used, with
labeled NAP conjugates added. Alternatively, the NAP conjugates can be
attached to the chip, either through the nucleic acid or through the protein
components of the system.
This may also be done using bead based systems; for example, for the
detection of nucleic acid binding proteins, standard "split and mix"
techniques, or
any standard oligonucleotide synthesis schemes, can be run using beads or
other
solid supports, such that libraries of sequences are made. The addition of NAP
conjugate libraries then allows for the detection of candidate proteins that
bind to
specific sequences.
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In some embodiments, only one of the components is labeled; alternatively,
more than one component may be labeled with different labels.
In a preferred embodiment, the binding of the candidate protein is
determined through the use of competitive binding assays. In this embodiment,
the competitor is a binding moiety known to bind to the target molecule such
as an
antibody, peptide, binding partner, ligand, etc. Under certain circumstances,
there
may be competitive binding as between the target and the binding moiety, with
the
binding moiety displacing the target.
Thus, a preferred utility of the invention is to determine the components to
which a drug will bind. That is, there are many drugs for which the targets
upon
which they act are unknown, or only partially known.
By starting with a drug, and NAP conjugates comprising a library of cDNA
expression products from the cell type on which the drug acts, the elucidation
of
the proteins to which the drug binds may be elucidated. By identifying other
proteins or targets in a signaling pathway, these newly identified proteins
can be
used in additional drug screens, as a tool for counterscreens, or to profile
chemically induced events. Furthermore, it is possible to run toxicity studies
using
this same method; by identifying proteins to which certain drugs undesirably
bind,
this information can be used to design drug derivatives without these
undesirable
side effects. Additionally, drug candidates can be run in these types of
screens to
look for any or all types of interactions, including undesirable binding
reactions.
Similarly, it is possible to run libraries of drug derivatives as the targets,
to
provide a two-dimensional analysis as well.
Positive controls and negative controls may be used in the assays.
Preferably all control and test samples are performed in at least triplicate
to obtain
statistically significant results. Incubation of all samples is for a time
sufficient for
the binding of the agent to the protein. Following incubation, all samples are
washed free of non-specifically bound material and the amount of bound,
generally labeled agent determined. For example, where a radiolabel is
employed,
the samples may be counted in a scintillation counter to determine the amount
of
bound compound. Similarly, ELISA techniques are generally preferred.
A variety of other reagents may be included in the screening assays. These
include reagents such as, but not limited to, salts, neutral proteins, e.g.
albumin,
detergents, etc which may be used to facilitate optimal protein-protein
binding
and/or reduce non-specific or background interactions. Also reagents that
otherwise improve the efficiency of the assay, such as protease inhibitors,
nuclease
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inhibitors, anti-microbial agents, co-factors such as cAMP, ATP, etc., may be
used. The mixture of components may be added in any order that provides for
the
requisite binding.
Screening for agents that modulate the activity of the target molecule may
5 also be done. As will be appreciated by those in the art, the actual screen
will
depend on the identity of the target molecule. In a preferred embodiment,
methods for screening for a candidate protein capable of modulating the
activity of
the target molecule comprise the steps of adding a NAP conjugate to a sample
of
the target, as above, and determining an alteration in the biological activity
of the
10 target. "Modulation" or "alteration" in this context includes an increase
in
activity, a decrease in activity, or a change in the type or kind of activity
present.
Thus, in this embodiment, the candidate protein should both bind to the target
(although this may not be necessary), and alter its biological or biochemical
activity as defined herein. The methods include both in vitro screening
methods,
15 as are generally outlined above, and ex vivo screening of cells for
alterations in
the presence, distribution, activity or amount of the target. Alternatively, a
candidate peptide can be identified that does not interfere with target
activity,
which can be useful in determining drug-drug interactions.
Thus, in this embodiment, the methods comprise combining a target
20 molecule and preferably a library of NAP conjugates and evaluating the
effect on
the target molecule's bioactivity. This can be done in a wide variety of ways,
as
will be appreciated by those in the art.
In these in vitro systems, e.g., cell-free systems, in either embodiment,
e.g.,
in vitro binding or activity assays, once a "hit" is found, the NAP conjugate
is
25 retrieved to allow identification of the candidate protein. Retrieval of
the NAP
conjugate can be done in a wide variety of ways, as will be appreciated by
those in
the art and will also depend on the type and configuration of the system being
used.
In a preferred embodiment, as outlined herein, a rescue tag or "retrieval
30 property" is used. As outlined above, a "retrieval property" is a property
that
enables isolation of the fusion enzyme when bound to the target. For example,
the
target can be constructed such that it is associated with biotin, which
enables
isolation of the target-bound fusion enzyme complexes using an affinity column
coated with streptavidin. Alternatively, the target can be attached to
magnetic
35 beads, which can be collected and separated from non-binding candidate
proteins
by altering the surrounding magnetic field. Alternatively, when the target
does not
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comprise a rescue tag, the NAP conjugate may comprise the rescue tag. For
example, affinity tags may be incorporated into the fusion proteins
themselves.
Similarly, the fusion enzyme-nucleic acid molecule complex can be also
recovered
by immunoprecipitation. Alternatively, rescue tags may comprise unique vector
sequences that can be used to PCR amplify the nucleic acid encoding the
candidate protein. In the latter embodiment, it may not be necessary to break
the
covalent attachment of the nucleic acid and the protein, if PCR sequences
outside
of this region (that do not span this region) are used.
In a preferred embodiment, after isolation of the NAP conjugate of interest,
the covalent linkage between the fusion enzyme and its coding nucleic acid
molecule can be severed using, for instance, nuclease-free proteases, the
addition
of non-specific nucleic acid, or any other conditions that preferentially
digest
proteins and not nucleic acids.
The nucleic acid molecules are purified using any suitable methods, such
as those methods known in the art, and are then available for further
amplification,
sequencing or evolution of the nucleic acid sequence encoding the desired
candidate protein. Suitable amplification techniques include all forms of PCR,
OLA, SDA, NASBA, TMA, Q-~3R, etc. Subsequent use of the information of the
"hit" is discussed below.
In a preferred embodiment, the NAP conjugates are used in ex vivo
screening techniques. In this embodiment, the expression vectors of the
invention
are introduced into host cells to screen for candidate proteins with a desired
property, e.g., capable of altering the phenotype of a cell. An advantage of
the
present inventive method is that screening of the fusion enzyme library can be
accomplished intracellularly. One of ordinary skill in the art will appreciate
the
advantages of screening candidate proteins within their natural environment,
as
opposed to lysing the cell to screen in vitro. In ex vivo or in vivo screening
methods, variant peptides are displayed in their native conformation and are
screened in the presence of other possibly interfering or enhancing cellular
agents.
Accordingly, screening intracellularly provides a more accurate picture of the
actual activity of the candidate protein and, therefore, is more predictive of
the
activity of the peptide ex vivo or in vivo. Moreover, the effect of the
candidate
protein on cellular physiology can be observed. Thus, the invention finds
particular use in the screening of eucaryotic cells.
Ex vivo and/or in vivo screening can be done in several ways. In a
preferred embodiment, the target need not be known; rather, cells containing
the
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expression vectors of the invention are screened for changes in phenotype.
Cells
exhibiting an altered phenotype are isolated, and the target to which the NAP
conjugate bound is identified as outlined below, although as will be
appreciated by
those in the art and outlined herein, it is also possible to bind the fusion
polypeptide and the target prior to forming the NAP conjugate. Alternatively,
the
target may be added exogeneously to the cell and screening for binding and/or
modulation of target activity is done. In the latter embodiment, the target
should
be able to penetrate the membrane, by, for instance, direct penetration or via
membrane transporting proteins, or by fusions with transport moieties such as
lipid moieties or HIV-tat, described below.
In general, experimental conditions allow for the formation of NAP
conjugates within the cells prior to screening, although this is not required.
That
is, the attachment of the NAM fusion enzyme to the EAS may occur at any time
during the screening, either before, during or after, as long as the
conditions are
1 S such that the attachment occurs prior to mixing of cells or cell lysates
containing
different fusion nucleic acids.
As will be appreciated by those in the art, the type of cells used in this
embodiment can vary widely. Basically, any eucaryotic or procaryotic cells can
be used, with mammalian cells being preferred, especially mouse, rat, primate
and
human cells. The host cells can be singular cells, or can be present in a
population
of cells, such as in a cell culture, tissue, organ, organ system, or organism
(e.g., an
insect, plant or animal). As is more fully described below, a screen will be
set up
such that the cells exhibit a selectable phenotype in the presence of a
candidate
protein. As is more fully described below, cell types implicated in a wide
variety
of disease conditions are particularly useful, so long as a suitable screen
may be
designed to allow the selection of cells that exhibit an altered phenotype as
a
consequence of the presence of a candidate agent within the cell.
Accordingly, suitable cell types include, but are not limited to, tumor cells
of all types (particularly melanoma, myeloid leukemia, carcinomas of the lung,
breast, ovaries, colon, kidney, prostate, pancreas and testes),
cardiomyocytes,
endothelial cells, epithelial cells, lymphocytes (T-cell and B cell) , mast
cells,
eosinophils, vascular intimal cells, hepatocytes, leukocytes including
mononuclear
leukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney, liver
and
myocyte stem cells (for use in screening for differentiation and de-
differentiation
factors), osteoclasts, chondrocytes and other connective tissue cells,
keratinocytes,
melanocytes, liver cells, kidney cells, and adipocytes. Suitable cells also
include
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known research cells, including, but not limited to, Jurkat T cells, NIH3T3
cells,
CHO, Cos, etc. See the ATCC cell line catalog, hereby expressly incorporated
by
reference.
In one embodiment, the cells may be genetically engineered, that is,
contain exogeneous nucleic acid, for example, to contain target molecules.
In a preferred embodiment, a first plurality of cells is screened. That is,
the
cells into which the expression vectors are introduced are screened for an
altered
phenotype. Thus, in this embodiment, the effect of the candidate protein is
seen in
the same cells in which it is made; i.e. an autocrine effect.
By a "plurality of cells" herein is meant roughly from about 103 cells to 108
or 109, with from 106 to 10g being preferred. This plurality of cells
comprises a
cellular library, wherein generally each cell within the library contains a
member
of the NAP conjugate molecular library, i.e. a different candidate protein,
although
as will be appreciated by those in the art, some cells within the library may
not
contain an expression vector and some may contain more than one.
In a preferred embodiment, the expression vectors are introduced into a
first plurality of cells, and the effect of the candidate proteins is screened
in a
second or third plurality of cells, different from the first plurality of
cells, i.e.
generally a different cell type. That is, the effect of the candidate protein
is due to
an extracellular effect on a second cell; i.e. an endocrine or paracrine
effect. This
is done using standard techniques. The first plurality of cells may be grown
in or
on one media, and the media is allowed to touch a second plurality of cells,
and
the effect measured. Alternatively, there may be direct contact between the
cells.
Thus, "contacting" is functional contact, and includes both direct and
indirect. In
this embodiment, the first plurality of cells may or may not be screened.
If necessary, the cells are treated to conditions suitable for the expression
of the fusion nucleic acids (for example, when inducible promoters are used),
to
produce the candidate proteins.
Thus, the methods of the present invention preferably comprise introducing
a molecular library of fusion nucleic acids or expression vectors into a
plurality of
cells, thereby creating a cellular library. Preferably, two or more of the
nucleic
acids comprises a different nucleotide sequence encoding a different candidate
protein. The plurality of cells is then screened, as is more fully outlined
below, for
a cell exhibiting an altered phenotype. The altered phenotype is due to the
presence of a candidate protein.
By "altered phenotype" or "changed physiology" or other grammatical
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equivalents herein is meant that the phenotype of the cell is altered in some
way,
preferably in some detectable and/or measurable way. As will be appreciated in
the art, a strength of the present invention is the wide variety of cell types
and
potential phenotypic changes which may be tested using the present methods.
Accordingly, any phenotypic change which may be observed, detected, or
measured may be the basis of the screening methods herein. Suitable phenotypic
changes include, but are not limited to: gross physical changes such as
changes in
cell morphology, cell growth, cell viability, adhesion to substrates or other
cells,
and cellular density; changes in the expression of one or more RNAs, proteins,
lipids, hormones, cytokines, or other molecules; changes in the equilibrium
state
(i.e. half life) or one or more RNAs, proteins, lipids, hormones, cytokines,
or other
molecules; changes in the localization of one or more RNAs, proteins, lipids,
hormones, cytokines, or other molecules; changes in the bioactivity or
specific
activity of one or more RNAs, proteins, lipids, hormones, cytokines,
receptors, or
other molecules; changes in the secretion of ions, cytokines, hormones, growth
factors, or other molecules; alterations in cellular membrane potentials,
polarization, integrity or transport; changes in infectivity, susceptability,
latency,
adhesion, and uptake of viruses and bacterial pathogens; etc. By "capable of
altering the phenotype" herein is meant that the candidate protein can change
the
phenotype of the cell in some detectable and/or measurable way.
The altered phenotype may be detected in a wide variety of ways, as is
described more fully below, and will generally depend and correspond to the
phenotype that is being changed. Generally, the changed phenotype is detected
using, for example: microscopic analysis of cell morphology; standard cell
viability assays, including both increased cell death and increased cell
viability,
for example, cells that are now resistant to cell death via virus, bacteria,
or
bacterial or synthetic toxins; standard labeling assays such as fluorometric
indicator assays for the presence or level of a particular cell or molecule,
including
FACS or other dye staining techniques; biochemical detection of the expression
of
target compounds after killing the cells; etc.
The present methods have utility in, for example, cancer applications. The
ability to rapidly and specifically kill tumor cells is a cornerstone of
cancer
chemotherapy. In general, using the methods of the present invention, random
or
directed libraries (including cDNA libraries) can be introduced into any tumor
cell
(primary or cultured), and peptides identified which by themselves induce
apoptosis, cell death, loss of cell division or decreased cell growth. This
may be
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done de novo, or by biased randomization toward known peptide agents, such as
angiostatin, which inhibits blood vessel wall growth. Alternatively, the
methods
of the present invention can be combined with other cancer therapeutics (e.g.
drugs or radiation) to sensitize the cells and thus induce rapid and specific
5 apoptosis, cell death, loss of cell division or decreased cell growth after
exposure
to a secondary agent. Similarly, the present methods may be used in
conjunction
with known cancer therapeutics to screen for agonists to make the therapeutic
more effective or less toxic. This is particularly preferred when the
chemotherapeutic is very expensive to produce such as taxol.
10 In a preferred embodiment, the present invention finds use with assays
involving infectious organisms. Intracellular organisms such as mycobacteria,
listeria, salmonella, pneumocystis, yersinia, leishmania, T. cruzi, can
persist and
replicate within cells, and become active in immunosuppressed patients. There
are
currently drugs on the market and in development which are either only
partially
15 effective or ineffective against these organisms. Candidate libraries can
be
inserted into specific cells infected with these organisms (pre- or post-
infection),
and candidate proteins selected which promote the intracellular destruction of
these organisms in a manner analogous to intracellular "antibiotic peptides"
similar to magainins. In addition peptides can be selected which enhance the
cidal
20 properties of drugs already under investigation which have insufficient
potency
by themselves, but when combined with a specific peptide from a candidate
library, are dramatically more potent through a synergistic mechanism.
Finally,
candidate proteins can be isolated which alter the metabolism of these
intracellular
organisms, in such a way as to terminate their intracellular life cycle by
inhibiting
25 a key organismal event.
In a preferred embodiment, the compositions and methods of the invention
are used to detect protein-protein interactions, similar to the use of a two-
hybrid
screen. This can be done in a variety of ways and in a variety of formats. As
will
be appreciated by those in the art, this embodiment and others outlined herein
can
30 be run as a "one dimensional" analysis or "multidimensional" analysis. That
is,
one NAP conjugate library can be run against a single target or against a
library of
targets. Alternatively, more than one NAP conjugate library can be run against
each other.
In a preferred embodiment, the compositions and methods of the invention
35 are used in protein drug discovery, particularly for protein drugs that
interact with
targets on cell surfaces.
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In a preferred embodiment, as outlined above, the compositions and
methods of the invention are used to discover DNA or nucleic acid binding
proteins, using nucleic acids as the targets.
In a preferred embodiment, the compositions and methods of the invention
are used to screen for NAM enzymes with decreased toxicity for the host cells.
For example, Rep proteins of the invention can be toxic to some host cells.
The
present inventive methods can be used to identify or generate Rep proteins
with
decreased toxicity. In this particular embodiment, Rep variants or, in an
alternative, random peptides are used in the present inventive conjugates to
observe cell toxicity and binding affinity to an EAS.
With respect to EASs, the present inventive methods can also be utilized to
identify novel or improved EASs for use in the present inventive expression
vectors. An EAS for a particular NAM enzyme of interest can also be identified
using the present inventive method. Formation of covalent structure of NAM
enzyme and EAS can determined using suitable methods that are present in the
art,
e.g. those described in U.S. patent 5545529. In general, the candidate NAM
enzyme can be expressed using a variety of hosts, such as bacteria or
mammalian
cells. The expressed protein can then be tested with candidate DNA sequences,
such a library of fragments obtained from the genome from which the NAM
enzyme is cloned. Contacts between the NAM enzyme and with the library of
DNA fragments under appropriate conditions (suchas inclusion of cofactors)
allow
for the formation of covalent NAM enzyme-DNA conjugates. The mixture can
then be separated using a variety of techniques. The isolated bound nucleic
acid
sequences can then be identified and sequenced. These sequences can be tested
further via a variety of mutagenesis techniques. The confirmed sequence motif
can then be used an EAS.
In a preferred embodiment, the compositions and methods of the invention
are used in pharmacogenetic studies. For example, by building libraries from
individuals with different phenotypes and testing them against targets,
differential
binding profiles can be generated. Thus, a preferred embodiment utilizes
differential binding profiles of NAP conjugates to targets to elucidate
disease
genes, SNPs or proteins.
In a preferred embodiment, once a cell with an altered phenotype is
detected, the cell is isolated from the plurality which do not have altered
phenotypes. This may be done in any number of ways, as is known in the art,
and
will in some instances depend on the assay or screen. Suitable isolation
techniques
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include, but are not limited to, FACS, lysis selection using complement, cell
cloning, scanning by Fluorimager, expression of a "survival" protein, induced
expression of a cell surface protein or other molecule that can be rendered
fluorescent or taggable for physical isolation; expression of an enzyme that
changes a non-fluorescent molecule to a fluorescent one; overgrowth against a
background of no or slow growth; death of cells and isolation of DNA or other
cell vitality indicator dyes, etc.
In a preferred embodiment, as outlined above, the NAP conjugate is
isolated from the positive cell. This may be done in a number of ways. In a
preferred embodiment, primers complementary to DNA regions common to the
NAP constructs, or to specific components of the library such as a rescue
sequence, defined above, are used to "rescue" the unique candidate protein
sequence. Alternatively, the candidate protein is isolated using a rescue
sequence.
Thus, for example, rescue sequences comprising epitope tags or purification
sequences may be used to pull out the candidate protein, using
immunoprecipitation or affinity columns. In some instances, as is outlined
below,
this may also pull out the primary target molecule, if there is a sufficiently
strong
binding interaction between the candidate protein and the target molecule.
Alternatively, the peptide may be detected using mass spectroscopy. Once
rescued, the sequence of the candidate protein and fusion nucleic acid can be
determined. This information can then be used in a number of ways, i.e.,
genomic
databases.
For in vitro, ex vivo, and in vivo screening methods, once the "hit" has
been identified, the results are preferably verified. As will be appreciated
by those
in the art, there are a variety of suitable methods that can be used. In a
preferred
embodiment, the candidate protein is resynthesized and reintroduced into the
target cells, to verify the effect. This may be done using recombinant
methods,
e.g. by transforming naive cells with the expression vector (or modified
versions,
e.g. with the candidate protein no longer part of a fusion), or alternatively
using
fusions to the HIV-1 Tat protein, and analogs and related proteins, which
allows
very high uptake into target cells. See for example, Fawell et al., PNAS USA
91:664 ( 1994); Frankel et al., Cell 55:1189 ( 1988); Savion et al., J. Biol.
Chem.
256:1149 (1981); Derossi et al., J. Biol. Chem. 269:10444 (1994); and Baldin
et
al., EMBO J. 9:1511 (1990), all of which are incorporated by reference.
In addition, for both in vitro and ex vivo screening methods, the process
may be used reiteratively. That is, the sequence of a candidate protein is
used to
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generate more candidate proteins. For example, the sequence of the protein may
be the basis of a second round of (biased) randomization, to develop agents
with
increased or altered activities. Alternatively, the second round of
randomization
may change the affinity of the agent. Furthermore, if the candidate protein is
a
S random peptide, it may be desirable to put the identified random region of
the
agent into other presentation structures, or to alter the sequence of the
constant
region of the presentation structure, to alter the conformation/shape of the
candidate protein.
The methods of using the present inventive library can involve many
rounds of screenings in order to identify a nucleic acid of interest. For
example,
once a nucleic acid molecule is identified, the method can be repeated using a
different target. Multiple libraries can be screened in parallel or
sequentially
and/or in combination to ensure accurate results. In addition, the method can
be
repeated to map pathways or metabolic processes by including an identified
candidate protein as a target in subsequent rounds of screening.
In a preferred embodiment, the candidate protein is used to identify target
molecules, i.e. the molecules with which the candidate protein interacts. As
will
be appreciated by those in the art, there may be primary target molecules, to
which
the protein binds or acts upon directly, and there may be secondary target
molecules, which are part of the signaling pathway affected by the protein
agent;
these might be termed "validated targets".
In a preferred embodiment, the candidate protein is used to pull out target
molecules. For example, as outlined herein, if the target molecules are
proteins,
the use of epitope tags or purification sequences can allow the purification
of
primary target molecules via biochemical means (co-immunoprecipitation,
affinity
columns, etc.). Alternatively, the peptide, when expressed in bacteria and
purified, can be used as a probe against a bacterial cDNA expression library
made
from mRNA of the target cell type. Or, peptides can be used as "bait" in
either
yeast or mammalian two or three hybrid systems. Such interaction cloning
approaches have been very useful to isolate DNA-binding proteins and other
interacting protein components. The peptides) can be combined with other
pharmacologic activators to study the epistatic relationships of signal
transduction
pathways in question. It is also possible to synthetically prepare labeled
peptides
and use it to screen a cDNA library expressed in bacteriophage for those cDNAs
which bind the peptide.
Once primary target molecules have been identified, secondary target
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molecules may be identified in the same manner, using the primary target as
the
"bait". In this manner, signaling pathways may be elucidated. Similarly,
protein
agents specific for secondary target molecules may also be discovered, to
allow a
number of protein agents to act on a single pathway, for example for
combination
therapies.
In a preferred embodiment, the methods and compositions of the invention
can be performed using a robotic system. Many systems are generally directed
to
the use of 96 (or more) well microtiter plates, but as will be appreciated by
those
in the art, any number of different plates or configurations may be used. In
addition, any or all of the steps outlined herein may be automated; thus, for
example, the systems may be completely or partially automated.
A wide variety of automatic components can be used to perform the
present inventive method or produce the present inventive compositions,
including, but not limited to, one or more robotic arms; plate handlers for
the
positioning of microplates; automated lid handlers to remove and replace lids
for
wells on non-cross contamination plates; tip assemblies for sample
distribution
with disposable tips; washable tip assemblies for sample distribution; 96 well
loading blocks; cooled reagent racks; microtiter plate pipette positions
(optionally
cooled); stacking towers for plates and tips; and computer systems.
Fully robotic or microfluidic systems include automated liquid-, particle-,
cell- and organism-handling including high throughput pipetting to perform all
steps of screening applications. This includes liquid, particle, cell, and
organism
manipulations such as aspiration, dispensing, mixing, diluting, washing,
accurate
volumetric transfers; retrieving, and discarding of pipet tips; and repetitive
pipetting of identical volumes for multiple deliveries from a single sample
aspiration. These manipulations are cross-contamination-free liquid, particle,
cell,
and organism transfers. This instrument performs automated replication of
microplate samples to filters, membranes, and/or daughter plates, high-density
transfers, full-plate serial dilutions, and high capacity operation.
In a preferred embodiment, chemically derivatized particles, plates, tubes,
magnetic particle, or other solid phase matrix with specificity to the assay
components are used. The binding surfaces of microplates, tubes or any solid
phase matrices include non-polar surfaces, highly polar surfaces, modified
dextran
coating to promote covalent binding, antibody coating, affinity media to bind
fusion proteins or peptides, surface-fixed proteins such as recombinant
protein A
or G, nucleotide resins or coatings, and other affinity matrix are useful in
this
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invention.
In a preferred embodiment, platforms for mufti-well plates, mufti-tubes,
minitubes, deep-well plates, microfuge tubes, cryovials, square well plates,
filters,
chips, optic fibers, beads, and other solid-phase matrices or platform with
various
volumes are accommodated on an upgradable modular platform for additional
capacity. This modular platform includes a variable speed orbital shaker,
electroporator, and mufti-position work decks for source samples, sample and
reagent dilution, assay plates, sample and reagent reservoirs, pipette tips,
and an
active wash station.
10 In a preferred embodiment, thermocycler and thermoregulating systems are
used for stabilizing the temperature of the heat exchangers such as controlled
blocks or platforms to provide accurate temperature control of incubating
samples
from 4 0 C to 100°C.
In a preferred embodiment, Interchangeable pipet heads (single or multi-
15 channel ) with single or multiple magnetic probes, affinity probes, or
pipetters
robotically manipulate the liquid, particles, cells, and organisms. Mufti-well
or
mufti-tube magnetic separators or platforms manipulate liquid, particles,
cells, and
organisms in single or multiple sample formats.
In some preferred embodiments, the instrumentation will include a
20 detector, which can be a wide variety of different detectors, depending on
the
labels and assay. In a preferred embodiment, useful detectors include a
microscopes) with multiple channels of fluorescence; plate readers to provide
fluorescent, ultraviolet and visible spectrophotometric detection with single
and
dual wavelength endpoint and kinetics capability, fluorescence resonance
energy
25 transfer (FRET), luminescence, quenching, two-photon excitation, and
intensity
redistribution; CCD cameras to capture and transform data and images into
quantifiable formats; and a computer workstation. These will enable the
monitoring of the size, growth and phenotypic expression of specific markers
on
cells, tissues, and organisms; target validation; lead optimization; data
analysis,
30 mining, organization, and integration of the high-throughput screens with
the
public and proprietary databases.
These instruments can fit in a sterile laminar flow or fume hood, or are
enclosed, self contained systems, for cell culture growth and transformation
in
mufti-well plates or tubes and for hazardous operations. The living cells will
be
35 grown under controlled growth conditions, with controls for temperature,
humidity, and gas for time series of the live cell assays. Automated
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transformation of cells and automated colony pickers will facilitate rapid
screening of desired cells.
Flow cytometry or capillary electrophoresis formats can be used for
individual capture of magnetic and other beads, particles, cells, and
organisms.
The flexible hardware and software allow instrument adaptability for
multiple applications. The software program modules allow creation,
modification, and running of methods. The system diagnostic modules allow
instrument alignment, correct connections, and motor operations. The
customized
tools, labware, and liquid, particle, cell and organism transfer patterns
allow
different applications to be performed. The database allows method and
parameter storage. Robotic and computer interfaces allow communication between
W struments.
In a preferred embodiment, the robotic workstation includes one or more
heating or cooling components. Depending on the reactions and reagents, either
cooling or heating may be required, which can be done using any number of
known heating and cooling systems, including Peltier systems.
In a preferred embodiment, the robotic apparatus includes a central
processing unit which communicates with a memory and a set of input/output
devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus. The
general
interaction between a central processing unit, a memory, input/output devices,
and
a bus is known in the art. Thus, a variety of different procedures, depending
on
the experiments to be run, are stored in the CPU memory.
The above-described methods of screening a pool of fusion enzyme-nucleic
acid molecule complexes for a nucleic acid encoding a desired candidate
protein
are merely based on the desired target property of the candidate protein. The
sequence or structure of the candidate proteins does not need to be known. A
significant advantage of the present invention is that no prior information
about
the candidate protein is needed during the screening, so long as the product
of the
identified coding nucleic acid sequence has biological activity, such as
specific
association with a targeted chemical or structural moiety. The identified
nucleic
acid molecule then can be used for understanding cellular processes as a
result of
the candidate protein's interaction with the target and, possibly, any
subsequent
therapeutic or toxic activity.
EXAMPLES
The following examples serve to more fully describe the manner of using
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the above-described invention, as well as to set forth the best modes
contemplated
for carrying out various aspects of the invention. It is understood that these
examples in no way serve to limit the true scope of this invention, but rather
are
presented for illustrative purposes.
Example 1
This example demonstrates the binding of an expressed fusion protein to its
coding nucleic acid molecule.
Plasmid pML2000, encoding a recombinant Rep78 - coding DNA fusion
fragment, was constructed using methods known in the art (see, for example,
Sambrook et al., supra). The plasmid, pML200 contained the following features:
a DNA replication origin functional in E.coli; an SV40 replication origin
functional in mammalian cells; a constitutive promoter that is active in the
host
cells, specifically the CMV promoter; and one copy of the AAV serotype 2
1 S inverted terminal repeat (ITR) sequence. The orientation of the ITR in
reference
to other components was not significant. The nucleic acid sequence that was
the
source of the AAV ITR had the sequence: 5'-
AGGAACCCCTAGTGATGGAGTTGGCCACT
CCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAA
AGCCCGGGCG - 3'. The duplex of the ITR sequence was previously shown to
be sufficient for interaction with a variant of Rep68 (Chiorini et al., 1994,
supra).
The resultant plasmid DNA was amplified in E.coli and purified using a
DNA maxiprep kit (Promega Inc., WI). The purified DNA was transfected into
tissue cultured HEK 293 cells (ATCC, MD) via calcium phosphate precipitation
or electroporation techniques. At 48 hours post-transfection, the cells were
harvested and lysed with 1% of Triton X-100 in standard phosphate buffered
saline (PBS). After centrifugation at 5000 x g for 30 minutes, the supernatant
was
used for subsequent biochemical characterization.
Expression of pML2000 in host cells allows for (i) expression of the
modified Rep78 protein as a fusion protein with a referenced partner, and (ii)
covalent attachment of the fusion protein to the attachment signal in a viral
or
plasmid vector. The expression of recombinant eREP was determined by
immunoblot analyses using either anti-HA antibody or anti-REP antibody. The
specific antibody binding was visualized by ECL chemiluminescence system
(Amersham-Pharmacia Biotech, IN). Expression of functional Rep78 proteins
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was previously demonstrated in the mammalian cell culture system (Li et al.,
J.
Virol., 71, 5236-5243 ( 1997)).
The ability to form DNA-eREP complexes was tested by the following
experiments. Host cells were transfected with two plasmids, pML2000 and
pML2000(DITR), individually and in combination. For each of the referenced
transfections, a total of 10 pg DNA was added in order to achieve a similar
level
of eREP protein expression. At 48 hours after transfection, the cells were
harvested and protein lysates were prepared. To test covalent binding between
the
expressed eREP and the plasmid DNA, the lysates were first boiled for 5
minutes
and immediately chilled on ice. An aliquot of boiled lysate from each sample
was
mixed with anti-REP antibody followed by incubation with an excess amount of
protein A agarose (Sigma, MO). After an extensive wash, the protein A agarose
beads were transferred to PCR tubes. The presence of bound plasmid was tested
by polymerase chain reaction to amplify the regions specific for either
plasmid.
The transfected plasmid pML2000 was precipitated by protein A agarose while
the pML2000 (DITR) was not precipitated. The formed eREP-pML2000 complex
was heat-resistant, consistent with the covalent bonding between eREP and the
expression plasmid pML2000. Furthermore, the interaction is ITR sequence-
specific similar to previous iri vitro and in vivo data (Yang et al., J.
Virol., 66,
6058-6069, (1992); Chiorini et al., J. Virol., 68, 797-804 (1994)).
This example demonstrates the construction of a vector suitable for use in
the present inventive methods. The results demonstrate that enzyme-vector
complexes are formed following expression of the Rep protein, and that binding
of
the Rep protein to its coding vector is covalent.
Example 2
The following example demonstrates a method of identifying and isolating
a nucleic acid molecule encoding a gene product comprising a target property
using an affinity column.
To retrieve a protein with a desired property, a chemical moiety, for
example, FK506 (CalBiochem Inc., CA) was purchased and chemically attached
to biotin using a commercial chemical linkage reagent. After conjugation, the
compound was purified via standard chromatographic techniques and confirmed
by NMR. To immobilize the compound, immobilon-4 96-well plates first were
coated with 10 q.g/ml streptavidin (SA). Following the coating, the
biotinylated-
FK506 in PBS was added to saturate all binding sites. After removal of the
excess
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biotinylated-FK506, the coated wells then were blocked with 1% BSA in PBS.
After washing, the immobilized compound was ready for affinity selection.
A library of lysates comprising fusion enzyme-expression vector
complexes were prepared by first transfecting approximately 10g mammalian HEK
cells with cDNA libraries prepared from mouse RNA using routine molecular
biology techniques. At 48 hours post-transfection, the cells were harvested
and
collected by centrifugation. The cells were lysed in the presence of
proteinase
inhibitors by the lysis procedures described in Example 1. The clarification
of
total crude lysate was carried out by centrifugation at 5000 x g for 30
minutes.
The prepared cell lysates were either stored at -80 °C or immediately
used with
immobilon-4 wells coated with biotinylated-FK506. After incubation with the
biotinylated-FK506, the lysate was removed from the immobilon-4 plates. The
wells were then washed extensively with PBS using the 12 well Nunc hand-held
washer (Corning, NY). The bound fusion enzyme-expression vector complexes
were released from the biotinylated-FK506 by incubation with 1 % trypsin. The
recovered DNA was extracted twice with Tris-buffered phenol and precipitated
using a standard ethanol precipitation procedure in the presence of 1 q.g of
glycogen. The precipitated DNA was washed once with 70% ethanol and
transformed into bacteria using electroporation. The isolated DNA can be
further
subjected to further rounds of affinity selection as desired.
This example demonstrates the isolation of a nucleic acid encoding a
peptide comprising a desired property, the ability to bind FK506, using the
methods of the present invention.
Example 3
The following example demonstrates a method of characterizing the cDNA
fragment inserted into the expression vector to form a fusion enzyme library.
cDNA encoding peptides with desired properties can be characterized by
employing ELISA procedures using standard protocols and antibodies specific
for
the NAM enzyme, e.g., Rep78. Thus, if a cDNA clone encodes a peptide that
interacts with FK506, it is expected that the cell lysate comprising the
referenced
plasmid DNA will be specific to FK506 coated wells, but not streptavidin (SA)-
coated or other negative control coated wells. Similarly, one expects that a
control
plasmid does not result in lysates that induce any ELISA signal.
After two rounds of affinity panning, performed as described in Example 2,
individual colonies of bacterial transformants were randomly selected.
Overnight
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cultures from single colonies in 3 ml of LB ampicillin ( 100 g,g/ml) were used
to
isolate DNA using a standard miniprep DNA kit (Promega, WI). Expression of
the eREP- variant peptide fusion proteins was achieved by transient
transfection
into HEK 293 cells. At 48 hours posttransfection, cell lysates were prepared
as
5 described in Example 2. Clarified lysates were used immediately for ELISA or
stored at -70° C. To prepare ELISA, 96-well plates were first coated
with SA
alone or SA + biotin-FK506. The wells were then blocked with 1% BSA in
phosphate buffered saline (PBS) at pH 7.4. After precoating with SA, the wells
were washed three times with PBS supplemented with 0.05% Tween-20 (PBT).
10 To initiate binding of the fusion enzyme-expression vector complexes to the
well
surface, 100 p.1 of 1:10 diluted lysate was added to each well. After 60
minutes at
4° C, the plates were washed four times with PBT. The binding of the
eREP
DNA-binding portion peptide of the fusion enzyme was detected using rabbit
anti-
REP antibody. After 4 washes with PBT, the plate was developed by adding
15 alkaline phosphatase-conjugated goat anti-rabbit antibody (GIBCO-BRL, MD)
in
PBS / 0.1 % BSA ( 100 ~1 per well for 1 hr at 25° C) followed by a 6 to
100-min
treatment with p-nitrophenyl phosphate (4 mg/ml) in 1 M diethanolamine
hydrochloride, pH 9.8/0.24 mM MgCl2 (200 ~1 per well). Binding was quantified
by monitoring optical density (0.D.) at 405 nm on an E-max plate reader
20 (Molecular Devices Inc., CA). The negative controls consisted of wells
coated
with control glutathione S-transferase (GST) fusion or as otherwise indicated.
Control plasmids, e.g., plasmids not comprising the coding sequence for a
FK506-
binding peptide, did not induce a signal in the ELISA assay. Fusion enzymes
comprising a peptide with the target property, FK506 binding, were identified
via
25 the ELISA assay. All experiments were repeated at least once with similar
results.
This example demonstrates a method of using a fusion enzyme library to
identify a peptide comprising a desired activity and to identify a nucleic
acid
encoding a target function by virtue of the fusion enzyme-expression vector
linkage.
Example 4
The following example demonstrates a method of using a fusion enzyme
library to identify a DNA binding peptide, the nucleic acid molecule encoding
the
DNA binding peptide, and the nucleic acid sequence recognized by the DNA
binding peptide.
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A fusion enzyme library is constructed as described in Example 1. A
population of random DNA sequences is generated to provide the DNA binding
substrate for the DNA binding peptide encoded by the fusion enzyme library.
DNA synthesis resin (bead) is used to make a lead oligonucleotide of 25 bases
(cassette I) containing a Not I restriction enzyme site. After synthesis, the
resin is
divided into four aliquots and allowed to proceed to the next step of
synthesis,
wherein an A, T, G, or C is added (each aliquot has a different base type
added).
After one cycle, the resin is mixed and divided into four aliquots for the
subsequent cycle, in which another A, T, G, or C is added individually to each
aliquot. The referenced mixing and dividing steps are repeated twelve times to
generate l2mer random oligonucleotide cassettes (ROC). The resin is then
mixed,
and an additional 20 base cassette is added (cassette II). The split-mix
synthesis
procedures allow for the generation of random oligonucleotide DNA fragments
wherein the resin mixture has "one sequence per bead." In other words, onto
each
1 S bead is attached many copies of a single oligonucleotide.
To obtain double stranded DNA binding substrate, the resultant resin mix
is washed with a buffer for Klenow enzyme. The washed resins are mixed with
synthetic oligonucleotides and an extension primer that is complementary to
cassette II. The mixture is heated to 80 °C, slowly cooled to 25
°C, and chilled to
4 °C, which allows the extension primer to hybridize to the template.
The
resultant mixture of resins is incubated in Klenow buffer under standard
conditions in the presence of dNTPs, such that an extension reaction is
carried out.
The resultant resin with double stranded DNA is then washed with standard PBS
buffer and stored at 4 °C in the presence of sodium azide.
To identify genes or coding sequences for DNA binding proteins, the resins
with attached DNA fragments are incubated with the fusion enzyme library
encoding putative DNA binding peptides at 4 °C for 12 hours. The bead-
REP
fusion enzyme complexes are marked with a primary antibody directed against
REP. Following the incubation, the mixture is incubated with magnetic beads
comprising pre-conjugated secondary antibody. After incubation, the bead-resin
mixture is heated to denature the protein and disconnect the magnetic bead -
oligonucleotide resin complexes. The magnetic beads are removed using standard
procedures, thereby isolating the co-precipitated non-magnetic DNA-resin. This
material can be used for PCR amplification and sequencing analyses either as a
pool or via single bead analyses procedures. Optionally, the resultant mixture
is
pelleted by centrifugation at 5000 x g for 10 minutes and washed extensively
with
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PBS. The bound protein-cDNA complexes on the resin are treated with proteinase
K. The nucleic acids coding for the desired fusion enzyme are recovered by
standard DNA preparation procedures. If desired, the recovered plasmids are
introduced into mammalian hosts and used for the subsequent rounds) of
affinity
selection. The binding sequences recognized by the DNA binding peptide can be
determined by sequencing PCR products of bound DNA to a particular NAM
enzyme-DNA binding peptide fusion. The DNA binding peptide can be identified
using protein analysis methods known in the art.
Collectively, the methods used herein allow for the generation of a series of
cDNAs encoding DNA binding proteins and their corresponding binding
sequences. For example, once a binding sequence has been identified using
random oligonucleotides, a homology search can be carried out to determine all
candidate sites in the human genome that represent possible binding sites for
a
given DNA binding protein. Conceivably, an integrated protein-DNA interaction
map/database for the human genome then can be generated.
All of the references cited herein, including patents, patent applications,
and
publications, are hereby incozporated in their entireties by reference.
While this invention has been described with an emphasis upon preferred
embodiments, variations of the preferred embodiments can be used, and it is
intended that the invention can be practiced otherwise than as specifically
described
herein. Accordingly, this invention includes all modifications encompassed
within
the spirit and scope of the invention as defined by the following claims.
