Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
PROTEIN FRAGMENT COMPLEMENTATION ASSAYS TO DETECT BIOMOLECULAR INTERACTIONS
FIELD OF THE INVENTION
The present invention relates to the determination of the
function of novel gene products. The invention further relates to Protein
fragment Complementation Assays (PCA). PCAs allow for the detection
of a wide variety of types of protein-protein, protein-RNA, protein-DNA,
Protein-carbohydrate or protein-small organic molecule interactions in
different cellular contexts appropriate to the study of such interactions.
BACKGROUND OF THE INVENTION
Many processes in biology, including transcription,
translation, and metabolic or signal transduction pathways, are mediated
by noN-covalently-associated multienzyme complexes1'101. The formation
of multiprotein or protein-nucleic acid complexes produce the most
efficient chemical machinery. Much of modern biological research is
concerned with identifying proteins involved in cellular processes,
determining their functions and how, when, and where they interact with
other proteins involved in specific pathways. Further, with rapid
advances in genome sequencing projects there is a need to develop
strategies to define "protein linkage maps", detailed inventories of protein
interactions that make up functional assemblies of proteins". Despite the
importance of understanding protein assembly in biological processes,
there are few convenient methods for studying protein-protein interactions
in vivo4.5. Approaches include the use of chemical crosslinking reagents
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and resonance energy transfer between dye-coupled proteins102 103. A
powerful and commonly used strategy, the yeast two-hybrid system, is
used to identify novel protein-protein interactions and to examine the
amino acid determinants of specific protein interactions4.8. The approach
allows for rapid screening of a large number of clones, including cDNA
libraries. Limitations of this technique include the fact that the interaction
must occur in a specific context (the nucleus of S. cerevisiae), and
generally cannot be used to distinguish induced versus constitutive
interactions.
Recently, a novel strategy for detecting protein-protein
interactions has been demonstrated by Johnsson and Varshavsky1 8
called the ubiquitin-based split protein sensor (USPS)9. The strategy is
based on cleavage of proteins with N-terminal fusions to ubiquitin by
cytosolic proteases (ubiquitinases) that recognize its tertiary structure.
The strategy depends on the reassembly of the tertiary structure of the
protein ubiquitin from complementary N- and C-terminal fragments and
crucially, on the augmention of this reassembly by oligomerization
domains fused to these fragments. Reassembly is detected as specific
proteolysis of the assembled product by cytosolic proteases
(ubiquitinases). The authors demonstrated that a fusion of a reporter
protein-ubiquitin C-terminal fragment could also be cleaved by
ubiquitinases, but only if co-expressed with an N-terminal fragment of
ubiquitin that was complementary to the C-terminal fragment. The
reconstitution of observable ubiquitinase activity only occurred if the N-
and C-terminal fragments were bound through GCN4 leucine
zippers109,110. The authors suggested that this "split-gene" strategy could
be used as an in vivo assay of protein-protein interactions and analysis
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of protein assembly kinetics in cells. Unfortunately, this strategy requires
additional cellular factors (in this case ubiquitinases) and the detection
method does not lend itself to high-throughput screening of cDNA
libraries. =
Rossi, F., C. A. Charlton, and H. M. Blau (1997) Proc.
Nat. Acad. Sci. (USA) 94, 8405-8410) have reported an assay based on
the classical complementation of a and w fragments of b-galactosidase
(b-gal) and induction of complementation by induced oligomerization of
the proteins FKBP12 and the mamalian target of rapamycin by rapamycin
in transfected C2C12 myoblast cell lines. Reconstitution of b-gal activity
is detected using substrate fluorescein di-b-D-galactopyranoside using
several fluorecence detection assays. While this assay bears some
resemblance to the present invention, there are several significant
distinguishing differences. First, this particular complementation
approach has been used for over thirty years in a vast number of
applications including the detection of protein-protein interactions.
Krevolin, M. and D. Kates (1993) U.S. Patent No. 5,362,625) teaches the
use of this complementation to detect protein-protein interactions. Also
achievement of b-gal complementation in mamalian cells has previously
been reported (Moosmann, P. and S. Rusconi (1996) Nucl. Acids Res.
24, 1171-1172). The individual PCAs presented here are completely de
novo designed interaction detection assays, not described in any way
previously except for publications arising from applicants laboratory.
Secondly, this application describes a general strategy to develop
molecular interaction assays from a large number of enzyme or protein
detectors, all de novo designed assays, whereas the b-gal assay is not
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novel, nor are any general strategies or advancements over previosly well
documented applications given.
As in the USPS, the yeast-two hybrid strategy requires
additional cellular machinery for detection that exist only in specific
cellular compartments. There is therefore a need for a detection system
which uses the reconstitution of a specific enzyme activity from fragments
as the assay itself, without the requirement for other proteins for the
detection of the activity. Preferably, the assay would involve an
oligomerization-assisted complementation of fragments of monomeric or
multimeric enzymes that require no other proteins for the detection of
their activity. Furthermore, if the structure of an enzyme were known it
would be possible to design fragments of the enzyme to ensure that the
reassembled fragments would be active and to introduce mutations to
alter the stringency of detection of reassembly. However, knowledge of
structure is not a prerequesite to the design of complementing fragments,
as will be explained below. The flexibility allowed in the design of such
an approach would make it applicable to situations where other detection
systems may not be suitable.
led to rapid progress in the identification of novel genes. In applications
Recent advances in human genomics research has
to biological and pharmaceutical research, there is now the pressing need
to determine the functions of novel gene products; for example, for genes
shown to be involved in disease phenotypes. It is in addressing
questions of function where genomics-based pharmaceutical research
becomes bogged down and there is now the need for advances in the
development of simple and automatable functional assays. A first step
in defining the function of a novel gene is to determine its interactions with
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other gene products in an appropriate context; that is, since proteins
make specific interactions with other proteins or other biopolymers as part
of functional assemblies, an appropriate way to examine the function of
a novel gene is to determine its physical relationships with the products
of other genes.
Screening techniques for protein interactions, such as
the yeast "two-hybrid" system, have transformed molecular biology, but
can only be used to study specific types of constitutively interacting
proteins or interactions of proteins with other molecules, in narrowly
defined cellular and compartmental contexts and require a complex
cellular machinery (transcription) to work. To rationally screen for protein
interactions within the context of a specific problem requires more flexible
approaches. Specifically, assays that meet criteria necessary not only
to detecting molecular interactions, but also to validating these
interactions as specific and biologically relevant.
A list of assay characteristics that meet such criteria are
as follows:
1) Allow for the detection of protein-protein, protein-DNA/RNA or protein-
drug interactions in vivo or in vitro.
2) Allow for the detection of these interactions in appropriate contexts,
such as within a specific organism, cell type, cellular compartment, or
organelle.
3) Allow for the detection of induced versus constitutive protein-protein
interactions (such as by a cell growth or inhibitory factor).
4) To be able to distinguish specific versus non-specific protein-protein
interactions by controlling the sensitivity of the assay.
5) Allow for the detection of the kinetics of protein assembly in cells.
- -
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6) Allow for screening of cDNA, small organic molecule, or DNA or RNA
libraries for molecular interactions.
SUMMARY OF THE INVENTION
The present invention seeks to provide the above-
mentioned needs for which the prior art is silent. The present invention
provides a general strategy for detecting protein interactions with other
biopolymers including other proteins, nucleic acids, carbohydrates or for
screening small molecule libraries for compounds of potential therapeutic
value. In a preferred embodiment, the instant invention seeks to provide
an oligomerization-assisted complementation of fragments of monomeric
enzymes that require no other proteins for the detection of their activity.
In one such embodiment, a protein-fragment complementation assay
(PCA) based on reconstitution of dihydrofolate reductase activity by
complementation of defined fragments of the enzyme in E. coil is
hereby provided. This assay requires no additional endogenous factors
for detecting specific protein-protein interactions (i.e. leucine zipper
interactions) and can be conveniently extended to screening cDNA,
nucleic acid, small molecule or protein design libraries for molecular
interactions. In addition, the assay can also be adapted for detection of
protein interactions in any cellular context or compartment and be used
to distinguish between induced versus constitutive protein interactions in
both prokaryotic and eukaryotic systems.
One particular strategy for designing a protein
complementation assay (PCA) is based on using the following
characteristics: 1) A protein or enzyme that is relatively small and
monomeric, 2) for which there is a large literature of structural and
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functional information, 3) for which simple assays exist for the
reconstitution of the protein or activity of the enzyme, both in vivo and in
vitro, and 4) for which overexpression in eukaryotic and prokaryotic cells
has been demonstrated. If these criteria are met, the structure of the
enzyme is used to decide the best position in the polypeptide chain to
split the gene in two, based on the following criteria: 1) The fragments
should result in subdomains of continuous polypeptide; that is, the
resulting fragments will not disrupt the subdomain structure of the protein,
2) the catalytic and cofactor binding sites should all be contained in one
fragment, and 3) resulting new N- and C-termini should be on the same
face of the protein to avoid the need for long peptide linkers and allow for
studies of orientation-dependence of protein binding.
It should be understood that the above mentioned
criteria do not all need to be satisfied for a proper working of the present
invention. It is an advantage that the enzyme be small, preferably
between 10-40 kDa. Although monomeric enzymes are preferred,
multimeric enzymes can also be envisaged as within the scope of the
present invention. The dimeric protein tyrosinase can be used in the
instant assay. The information on the structure of the enzyme provides an
additional advantage in designing the PCA, but is not necessary. Indeed,
an additional strategy, to develop PCAs is presented, based on a
combination of exonuclease digestion-generated protein fragements
followed by directed protein evolution in application to the enzyme
aminoglycoside kinase. Although the overexpression in prokaryotic cells
is preferred it is not a necessity. It will be understood to the skilled
artisan
that the enzyme catalytic site (of the chosen enzyme) does not absolutely
need to be on same molecule.
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The present application explains the rationale and
criteria for using a particular enzyme in a PCA. Figure 1 shows a
general description of a PCA. The gene for a protein or enzyme is
rationally dissected into two or more fragments. Using molecular biology
techniques, the chosen fragments are subcloned, and to the 5' ends of
each, proteins that either are known or thought to interact are fused. Co-
transfection or transformation these DNA constructs into cells is then
carried out. Reassembly of the probe protein or enzyme from its
fragments is catalyzed by the binding of the test proteins to each other,
and reconstitution is observed with some assay. It is crucial to understand
that these assays will only work if the fused, interacting proteins catalyze
the reassembly of the enzyme. That is, observation of reconstituted
enzyme activity must be a measure of the interaction of the fused
proteins.
A preferred embodiment of the present invention
focuses on a PCA based on the enzyme dihydrofolate reductase.
Expansion of the strategy to include assays in eukaryotic, cells, library
screening, and a specific application to problems concerning the study of
integrated biochemical pathways such as signal transduction pathways,
is presented. Additional assays, including those based on enzymes that
can act as dominant or recesive drug selection or metabolic salvage
pathways are disclosed. In addition, PCAs based on enzymes that will
produce a colored or fluorescent product are also disclosed. The present
invention teaches how the PCA strategy can be both generalized and
automated for functional testing of novel genes, screening of natural
products or compound libraries for pharmacological activity and
identification of novel gene products that interact with DNA, RNA or
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carbohydrates are disclosed. It also teaches how the PCA strategy can
be applied to identifying natural products or small molecules from
compound libraries of potential therapeutic value that can inhibit or
activate such molecular interactions and how enzyme substrates and
small molecule inhibitors of enzymes can be identified. Finally, it teaches
how the PCA strategy can be used to perform protein engineering
experiments that could lead to designed enzymes with industrial
applications or peptides with biological activity.
Simple strategies to design and implement assays for
detecting protein interactions in vivo are disclosed herein. We have
designed complementary fragments of the native mDHFR that, when
coexpressed in E. coil grown in minimal medium, allow for survival of
clones expressing the two fragments, where the basal activity of the
endogenous bacterial DHFR is inhibited by the competitive inhibitor
trimethoprim (Fig. 3). Reconstitution of activity only occurred when both
N- and C-terminal fragments of DHFR were coexpressed as C-terminal
fusions to GCN4 leucine zipper sequences, indicating that reassembly of
the fragments requires formation of a leucine zipper between the N- and
C-terminal fusion peptides. The sequential increase in cell doubling
times resulting from the destabilizing mutations directed at the assembly
interface (1Ie114 to Val, Ala or Gly) demonstrates that the observed cell
survival under selective conditions is a result of the specific, leucine-
zipper-assisted association of mDHFR fragment[1,2] with fragment[3], as
opposed to nonspecific interactions of Z-F[3] with Z-F[1,2]. several
detailed and many additional examples are given.
As demonstrated previously with the ubiquitin-based
split protein sensor (USPS)9, a protein-fragment complementation
_
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strategy can be used to study equilibrium and kinetic aspects of protein-
protein interactions in vivo. The DHFR and other PCAs however, are
simpler assays. They are complete systems; no additional endogenous
factors are necessary and the results of complementation are observed
directly, with no further manipulation. The E. coli cell survival assay
described herein should therefore be particularly useful for screening
cDNA libraries for protein-protein interactions. mDHFR expression in
cells can be monitored by binding of fluorescent high-affinity substrate
analogues for DHFR26.There are several further aspects of the PCAs that
distinguish them from all other strategies for studying protein-protein
interactions in vivo (except USPS). We have designed complementary
fragments of enzymes that allow for controlling the stringency of the
assay, and could be used to obtain estimates of the kinetics and
equilibrium constants for association of two proteins. For example, with
DHFR the point mutations of the wild-type enzyme Ile 114 to Val, Ala, or
Gly alter the stringency of reconstitution of DHFR activity. For
determining estimates of equilibrium and kinetic parameters for a specific
protein-protein interaction, one could. perform a series of DHFR PCA
experiments with two proteins that interact with a known affinity, using the
wild type or destabilizing mutant DHFR fragments. Comparison of cell
growth rates in this model system with rates for a DHFR PCA using
unknowns would give an estimate of the strength of the unknown
interaction.
It should be understood that the present invention
should not be limited to the DHFR or other PCAs presented, as it is only
non-limiting embodiments of the protein complementation assay of the
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present invention. Moreover, the PCAs should not be limited in the
context in which they could be used. Constructs could be designed for
targeting the PCA fusions to specific compartments in the cell by addition
of signaling peptide sequences". Induced versus constitutive protein-
protein interactions could be distinguished by a eukaryotic version of the
PCA, in the case of an interaction that is triggered by a biochemical
event. Also, the system could be adapted for use in screening for novel,
induced protein-molecular associations between a target protein and an
expression library. The instant
invention is also directed to a method for
detecting biomolecular interactions said method comprising: (a)
selecting an appropriate reporter molecule;
(b) effecting fragmentation of said reporter molecule
such that said fragmentation results in reversible loss of reporter function;
(c) fusing or attaching fragments of said reporter
molecule separately to other molecules; followed by (d)
reassociation of said reporter fragments through
interactions of the molecules that are fused to said fragments.
complementation assays for the detection of molecular interactions The
invention also provides molecular fragment
comprising a reassembly of separate fragments of a molecule, wherein
reassembly of said fragments is operated by the interaction of molecular
domains fused to each fragment of said molecules, and wherein
reassembly of the fragments is independent of other molecular
processes.
In another aspect, the present invention is directed to a
method of testing biomolecular interactions comprising:
_
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a) generating a first fusion product comprising
i) a first fragment of a first molecule and
ii) a second molecule which is different or the
same as said first molecule;
b) generating a second fusion product comprising
i) a second fragment of said first molecule; and
ii) a third molecule which is different from or the
same as said first molecule or second molecule;
C) allowing the first and second fusion products to
contact each other; and
d) testing for activity regained by association of the
recombined fragments of the first molecule, wherein said reassociation is
mediated by interaction of the second and third molecules.
In another novel feature, the invention is directed to a
method comprising an assay where fragments of a first molecule are
fused to a second molecule and fragment association is detected by
reconstitution of the first molecule's activity.
The present invention also provides a composition
comprising a product selected from the group consisting of:
(a) a first fusion product comprising:
1) a first fragment of a first molecule whose
fragments can exhibit a detectable activity when associated and
2) a second molecule that can bind (a)(1);
(b) a second fusion product comprising
1) a second fragment of said first molecule and
2) a third molecule that can bind (b)(1); and
c) both (a) and (b).
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The invention further provides a composition comprising
complementary fragments of a first molecule, each fused to a separate
fragment of a second molecule.
The inventors of the present subject matter further
provide a composition comprising a nucleic acid molecule coding for a
fusion product, which molecule comprises sequences coding for a
product selected from the group consisting of:
(a) a first fusion product comprising:
1) fragments of a first molecule whose fragments
can exhibit a detectable activity when associated and
2) a second molecule fused to the fragment of the
first molecule;
(b) a second fusion product comprising
1) a second fragment of said first molecule and
2) a second or third molecule; and
(c) both (a) and (b).
The present invention is also directed to a method of
testing for biomolecular interactions associated with: (a) complementary
fragments of a first molecule whose fragments can exhibit a detectable
activity when associated or (b) binding of two protein-protein interacting
domains from a second or third molecule, said method comprising:
1) creating a fusion of
(a) a first fragment of a first molecule whose
fragments can exhibit a detectable activity when associated and
(b) a first protein-protein interacting domain;
2) creating a fusion of
(a) a second fragment of said first molecule and
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(b) a second protein-protein interacting domain
that can bind said first protein-protein interacting domain;
3) allowing the fusions of (1) and (2) to contact each
other; and
4) testing for said activity.
The instant inevntion further provides a composition
comprising a product selected from the group consisting of:
(a) a first fusion product comprising:
1) a first fragment of a molecule whose fragments
can exhibit a detectable activity when associated and
2) a first protein-protein interacting domain;
(b) a second fusion product comprising
1) a second fragment of said first molecule and
2) a second protein-protein interacting domain
that can bind said first protein-protein interacting domain; and
(c) both (a) and (b).
The invention is also directed to a composition
comprising a nucleic acid molecule coding for a fusion product, which
molecule comprises sequences coding for either:
(a) a first fusion product comprising:
1) a first fragment of a molecule whose fragments
can exhibit a detectable activity when associated and
2) a first protein-protein interacting domain; or
(b) a second fusion product comprising
1) a second fragment of said molecule and
2) a second protein-protein interacting domain
that can bind said first protein-protein interacting domain; or
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(c) both (a) and (b).
The invention also provides a method of detecting
kinetics of protein assembly and screening cDNA libraries comprising
performing PCA.
In another embodiment, the invention further provides
a method of testing the ability of a compound to inhibit molecular
interactions in a PCA comprising performing a PCA in the presence of
said compound and correlating any inhibition with said presence.
In a further embodiment, the invention provides a
method for detecting protein-protein interactions in living organisms and
or cells, which method comprises:
(a) synthesizing probe protein fragments from an
enzyme which enables dominant selection by dissecting the gene coding
for the enzyme into at least two fragments;
(b) constructing fusion proteins with one or more
molecules that are to be tested for interactions;
(c) fusing the proteins obtained in (b) with one or more
of the probe fragments;
(d) coexpressing the fusion proteins; and
(e) detecting the reconstitution of enzyme activity.
The invention still provides a method for detecting
biomolecular interactions said method comprising:
(a) selecting an appropriate reporter molecule;
(b) effecting fragmentation of said reporter molecule;
(c) fusing or attaching fragments of said reporter
molecule separately to other molecules; followed by
=
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(d) reassociation of said reporter fragments through
interactions of the molecules that are fused to said fragments.
Lastly, the invention also provides a novel method of
affecting gene therapy, which includes the step of providing the assays
and compositions described above.
The present invention is pionneering as it is the first
protein complementation assay displaying such a level of simplicity and
versatility. The exemplified embodiments are protein-fragment
complementation assays (PCA) based on mDHFR, where a leucine
zipper directs the reconstitution of DHFR activity. Activity was detected
by an E. coli survival assay which is both practical and inexpensive. This
system illustrates the use of mDHFR fragment complementation in the
detection of leucine zipper dimerization and could be applied to the
detection of unknown, specific protein-molecular interactions in vivo.
It should be undertstood that the instant invention is not
limited to the PCAs presented here, as numerous other enzymes can be
selected and used in accordance with the teachings of the present
invention. Examples of such markers can be found in Kaufman, (1987
Genetic Eng. 9:155-198) and references found therein as well as table 1
of this application.
It should also be clear to the skilled artisan to which the
present invention pertains that the invention is not limited to the use of
leucine zippers as the two interacting molecules. Indeed, numerous other
types of protein-molecule interactions can be used and identified in
accordance with the teaching of the present invention. The known types
of motifs involved in protein-molecular interactions are well known in the
art.
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Other features and advantages of the present invention
will be apparent from the following description of the preferred
embodiments thereof, the appended Examples and from the enjoined
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides a general description of a PCA. Using
molecular biology techniques, the chosen fragments of the enzyme are
subcloned, and to the 5' ends of each, proteins that either are known or
thought to interact are fused. Co-transfection or transformation these
DNA constructs into cells is then carried out and reconstitution with some
assay is observed.
FIG. 2 is a scheme of the fusion constructs used in one
of the embodiments of the invention. The hexahistidine peptide (6His),
the homodimerizing GCN4 leucine zipper (Zipper) and mDHFR fragments
(1, 2 and 3) are illustrated. The labels for the constructs are used to
identify both the DNA constructs and the proteins expressed from these
constructs.
FIG. 3: (A) shows E. coil survival assay on minimal
medium plates. Control: Left side of the plate: E. coli harboring pQE-30
(no insert); right side: E. coil harboring pQE-16, coding for native mDHFR.
Panel I: Left side of each plate: transformation with construct Z-F[1,2];
right side of each plate: transformation with construct Z-F[3]. Panel II:
Cotransformation with constructs Z-F[1,2] and Z-F[3]. Panel III:
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Cotransformation with constructs Control-F[1 ,2] and Z-F[3]. All plates
contain 0.5 mg/mItrimethoprim. In panels Ito III, plates on the right side
contain 1mM IPTG.
= (B) E. coil survival assay using destabilizing DHFR
mutants. Panel 1: Cotransformation of E. coil with constructs Z-F[1,2] and
Z-F[3:11e114Val]. Panel 11: Cotransformation with Z-F[1,2] and Z-
F[3:11e114Ala]. Inset is a 5-fold enlargement of the right-side plate. Panel
III: Cotransformation with Z-F[1,2] and Z-F[3:11e114Gly]. All plates contain
0.5 mg/ml trimethoprim. Plates on the right side contain 1mM IPTG.
FIG. 4 features the coexpression of mDHFR fragments.
(A) Agarose gel analysis of restriction pattern resulting from Hincll
digestion of plasmid DNA. Lane 1 contains DNA isolated from E. coli
cotransformed with constructs Z-F[1,2] and Z-F[3]. Lanes 2 and 3 contain
DNA isolated from E. coli transformed with, respectively, construct Z-F[3]
and construct Z-F[1,2]. Fragment migration (in bp) is indicated to the
right.
(B) SDS-PAGE analysis of mDHFR fragment
expression. Lanes 1 to 5 show crude lysate of untransformed E. coli
(lane 1), or E. coil expressing Z-F[1,2] (20.8 kDa; lane 2), Z-F[3] (18.4
kDa; lane 3), Control-F[1,2] (14.2 kDa; lane 4), and Z-F[1,2] + Z-F[3] (lane
5). Lane 6 shows 40 ml out of 2m1 copurified Z-F[1,2] and Z-F[3].
Arrowheads point to the proteins of interest. Migration of molecular
weight markers (in kDa) is indicated to the right.
FIG. 5 illustrates the general features of a PCA based
on a survival assay such as the DHFR PCA. The assay can be used in
a bacterial or a mammalian context. The inserted target DNA can be a
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known sequence coding for a protein (or protein domain) of interest, or
can be a cDNA library.
FIG. 6 represents an autoradiograph of a COS cell
lysate after a 30 min. 'S-Met-Cys pulse-labelling. The expression pattern
is essentially identical to that observed in E. coil (see Fig. 4). The DNA
transfected into the cells (or cotransfected) is indicated above the
respective lanes.FIG. 7 illustrates the results of a protein engineering
application of the mDHFR bacterial PCA. Two semi-random leucine
zipper libraries were created (as described in the text) and each inserted
N-terminal to one of the mDHFR fragments. Cotransformation of the
resulting zipper-DHFR fragment libraries in E. co/land plating on selective
medium allowed for survival of clones harboring successfully interacting
leucine zippers. Fourteen clones were isolated and the zippers were
sequenced to identify the residues at the 6e6 and 6g6 positions. The
6e-g6 pairs were categorized, as having attractive pairing
(charge:charge, charge:neutral polar or neutral polar:neutral polar) or
repulsive pairing (charge:charge) and the number of each type of
interaction scored for each clone. The total number of interactions for
each clone is 6; the interactions are tallied on the histogram.
Other objects, advantages and features of the present
invention will become more apparent upon reading of the following non-
restrictive description of preferred embodiments with reference to the
accompanying drawings which are examplary and should not be
interpreted as limiting the scope of the present invention.
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DESCRIPTION OF THE PREFERRED EMBODIMENTS
Selection of mDHFR for a PCA
In designing a protein-fragment complementation assay
(PCA), we sought to identify an enzyme for which the following is true: 1)
An enzyme that is relatively small and monomeric, 2) for which structural
and functional information exists, 3) for which simple assays exist for both
in vivo and in vitro measurement, and 4) for which overexpression in
eukaryotic and prokaryotic cells has been demonstrated. Murine DHFR
(mDHFR) meets all of the criteria for a PCA listed above. Prokaryotic and
eukaryotic DHFR is central to cellular one-carbon metabolism and is
absolutely required for cell survival in both prokaryotes and eukaryotes.
Specifically it catalyses the reduction of dihydrofolate to tetrahydrofolate
for use in transfer of one-carbon units required for biosynthesis of serine,
methionine, purines and thymidylate. The DHFRs are small (17 kD to 21
kD), monomeric proteins. The crystal structures of DHFR from various
bacterial and eukaryotic sources are known and substrate binding sites
and active site residues have been determined 111-114, allowing for rational
design of protein fragments. The folding, catalysis, and kinetics of a
number of DHFRs have been studied extensively115-119. The enzyme
activity can be monitored in vitro by a simple spectrophotometric assay'',
or in vivo by cell survival in cells grown in the absence of DHFR end
products. DHFR is specifically inhibited by the anti-folate drug
trimethoprim. As mammalian DHFR has a 12000-fold lower affinity for
trimethoprim than does bacterial DHFR121, growth of bacteria expressing
mDHFR in the presence of trimethoprim levels lethal to bacteria is an
efficient means of selecting for reassembly of mDHFR fragments into
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active enzyme. High level expression of mDHFR has been demonstrated
in transformed prokaryote or transfected eukaryotic ce1ls122-126,
Design Considerations.
mDHFR shares high sequence identity with the human
DHFR (hDHFR) sequence (91% identity) and is highly homologous to the
E. coli enzyme (29% identity, 68% homology) and these sequences share
visually superimposable tertiary structure'. Comparison of the crystal
structures of mDHFR and hDHFR suggests that their active sites are
essentially identical127.128. DHFR has been described as being formed of
three structural fragments forming two domains129.1" the adenine binding
domain (residues 47 to 105 = fragment[2]) and a discontinuous domain
(residues 1 to 46 = fragment[1] and 106 to 186 [3]; numbering according
to the murine sequence). The folate binding pocket and the NADPH
binding groove are formed mainly by residues belonging to fragments[1]
and [2]. Fragment [3] is not directly implicated in catalysis.
Residues 101 to 108 of hDHFR, at the junction between
fragment[2] and fragment[3], form a disordered loop which lies on the
same face of the protein as both termini. We chose to cleave mDHFR
between fragments [1,2] and [3], at residue 107, so as to cause minimal
disruption of the active site and NADPH cofactor binding sites. The
native N- terminus of mDHFR and the novel N-terminus created by
cleavage occur on the same surface of the enzyme112, 128 allowing for ease
of N-terminal covalent attachment of each fragment to associating
fragments such as the leucine zippers used in this study. Using this
system, we have obtained leucine-zipper assisted assembly of the
mDHFR fragments into active enzyme.
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EXAMPLE 1
EXPERIMENTAL PROTOCOL
DNA Constructs
Mutagenic and sequencing oligonucleotides were
purchased from Gibco BRL. Restriction endonucleases and DNA
modifying enzymes were from Pharmacia and New England Biolabs. The
mDHFR fragments carrying their own iN-frame stop codon were
subcloned into pQE-32 (Qiagen), downstream from and iN-frame with the
hexahistidine peptide and a GCN4 leucine zipper (Fig 1; Fig. 2). All final
constructs were based on the Qiagen pQE series of vectors, which
contain an inducible promoter-operator element (tac), a consensus
ribosomal binding site, initiator codon and nucleotides coding for a
hexahistidine peptide. Full-length mDHFR is expressed from pQE-16
(Qiagen).
Expression vector harboring the GCN4 leucine zipper
Residues 235 to 281 of the GCN4 leucine zipper (a
Sall/BamH1 254 bp fragment) were obtained from a yeast expression
plasmid pRS3169. The recessed terminus at the BamHI site was filled-in
with Klenow polymerase and the fragment was ligated to pQE-32
linearized with Sall/Hind111(filled-in). The product, construct Z, carries an
open reading frame coding for the sequence Met-Arg-Gly-Ser followed by
a hexahistidine tag and 13 residues preceding the GCN4 leucine zipper
residues.
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Creation of DHFR fragments: The eukaryotic transient expression vector, pMT3
(derived from pMT2)16, was used as a template for PCR-generation of
mDHFR containing the features allowing subcloning and separate
expression of fragment[1,2] and fragment[3]. The megaprimer method of
PCR mutagenesis' was used to generate a full-length 590 bp product.
Oligonucleotides complementary to the nucleotide sequence coding for
the N- and C-termini of mDHFR and containing a novel BspEl site outside
the coding sequence were used as well as an oligonucleotide used to
create a novel stop codon after fragment[1,2], followed by a novel Spel
site for use in subcloning fragment[3].
Construction of a new multiple cloning region and subcloning of
DHFR fragments [1.2] and [31Complementary oligonucleotides containing the
novel
restriction sites: SnaBl, Nhel, Spel and BspEl, were hybridized together
resulting in 5' and 3' overhangs complementary to EcoRI, and inserted
into pMT3 at a unique EcoRI site. The 590 bp PCR product (described
above) was digested with BspEl and .inserted into pMT3 linearized at
BspEl, yielding construct [1,2,3]. The 610 bp BspEl/EcoNI fragment
(coding for DHFR fragment[1,2], followed by a novel stop and fragment[3]
up to EcoNI) was filled in at EcoNI and subcloned into pMT3 opened with
BspEl/Hpal, yielding construct F[1,2]. The 250 bp Spel/BspEl fragment
of construct [1,2,3] coding for DHFR fragment[3] (with no in-frame stop
codon) was subcloned into pMT3 opened with the same enzymes. The
stop codon of the wild-type DHFR sequence, downstream from
fragment[3] in pMT3, was inserted as follows. Cleavage with EcoNI,
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present in both the inserted fragment[3] and the wild-type fragment[3],
removal of the 683 bp intervening sequence and religation of the vector
yielded a construct of fragment[3] with the wild-type stop codon, construct
F[3].
Creation of the expression constructs
The 1051 bp and the 958 bp SnaBI/Xbal fragments of
constructs F[1,2] and F[3], respectively, were subcloned into construct Z
opened with Bg111(filled-in)/Nhel, yielding constructs Z-F[1,2] and Z-F[3]
(Fig. 2). For the Control expression construct, the 180 bp Xmal/BspEl
fragment coding for the zipper was removed from construct Z-F[1,2],
yielding construct Control-F[1,2] (Fig. 2).
Creation of Stability Mutants Site-directed mutagenesis was performed3 to
produce
mutants at 11e114 (numbering of the wild-type mDHFR). The mutagenesis
reaction was carried out on the Kpnl/BamH1 fragment of construct Z-F[3]
subcloned into pBluescript SK+ (Stratagene), using oligonucleotides that
encode a silent mutation producing a novel BamH1 site. The 206 bp
Nhel/EcoNI fragment of putative mutants identified by restriction was
subcloned back into Z-F[3]. The mutations were confirmed by DNA
sequencing.
E. con Survival Assay
E. coli strain BL21 carrying plasmid pRep4 (from
Qiagen, for constitutive expression of the lac repressor) were made
competent, transformed with the appropriate DNA constructs and washed
1
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twice with minimal medium before plating on minimal medium plates
containing 50 mg/ml kanamycin, 100 mg/ml ampicillin and 0.5 mg/ml
trimethoprim. One half of each transformation mixture was plated in the
absence, and the second half in the presence, of 1 mM IPTG. All plates
were placed at 37 C for 66 hrs.
E. con Growth Curves
Colonies obtained from cotransformation were
propagated and used to inoculate 10 ml of minimal medium
supplemented with ampicillin, kanamycin as well as 1PTG (1mM) and
trimethoprim (1 mg/ml) where indicated. Cotransformants of Z-F[1,2] +
Z-F[3:11e114Gly] were obtained under non-selective conditions by plating
the transformation mixture on L-agar (+ kanamycin and ampicillin) and
screening for the presence of the two constructs by restriction analysis.
All growth curves were performed in triplicate. Aliquots were withdrawn
periodically for measurement of optical density. Doubling time was
calculated for early logarithmic growth (OD 600 between 0.02 and 0.2).
Protein Overexpression and Purification
Bacteria were propagated in Terrific Broth' in the
presence of the appropriate antibiotics to an 0D600 of approximately 1Ø
Expression was induced by addition of 1 mM 1PTG and further incubation
for 3 hrs. For analysis of crude extract, pellets from 150 ml of induced
cells were lysed by boiling in loading dye. The lysates were clarified by
microcentrifugation and analyzed by SDS-PAGE32. For protein
purification, a cell pellet from 50 ml of induced E. coil cotransformed with
constructs Z-F[1,2] and Z-F[3] was lysed by sonication, and a denaturing
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purification of the insoluble pellet undertaken using Ni-NTA (Qiagen) as
described by the manufacturer. The proteins were eluted with a stepwise
imidazole gradient. The fractions were analyzed by SDS-PAGE.
RESULTS
Design of mDHFR fragments for a PCA
mDHFR shares high sequence identity with the human
DHFR (hDHFR) sequence. As the coordinates of the murine crystal
structure were not available, we based our design considerations on the
hDHFR structure. DHFR has been described as comprising three
structural fragments forming two domains: the adenine binding domain
(F[2]) and a discontinuous domain (F[1] and F[3])13.18. The folate binding
pocket and the NADPH binding groove are formed mainly by residues
belonging to F[1] and F[2]. Residues 101 to 108 of hDHFR form a
disordered loop which lies on the same face of the protein as both termini.
This loop occurs at the junction between F[2] and F[3]. By cleaving
mDHFR at residue 107, we created F[1,21 and F[3], thus causing minimal
disruption of the active site and substrate binding sites. The native N-
terminus of mDHFR and the novel N-terminus created by cleavage were
covalently attached to the C-termini of GCN4 leucine zippers (Fig. 1).
E. coil Survival Assays
Figure 2 illustrates the general features of the expressed
constructs and the nomenclature used in this study. Figure 3 (panel A)
illustrates the results of cotransformation of bacteria with constructs
coding for Z-F[1,2] and Z-F[3], in the presence of trimethoprim, clearly
showing that colony growth under selective pressure is possible only in
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cells expressing both fragments of mDHFR. There is no growth in the
presence of either Z-F[1,2] or Z-F[3] alone. Induction of protein
expression with IPTG is essential for colony growth (Fig. 3A). The
presence of the leucine zipper on both fragments of mDHFR is essential
as illustrated by cotransformation of bacteria with both vectors coding for
mDHFR fragments, only one of which carries a leucine zipper (Fig. 3A).
It should be noted that growth of control E. coli transformed with the full-
length mDHFR is possible in the absence of IPTG due to low levels of
expression in uninduced cells.
Confirmation of the presence of both plasmids in
bacteria able to grow with trimethoprim was obtained from restriction
analysis of the plasmid DNA purified from isolated colonies. Figure 4 (A)
reveals the presence of the 1200 bp Hincll restriction fragment from
construct Z-F[1,2] as well as the 487 and 599 bp Hincll restriction
fragments from construct Z-F[3]. Also present is the 935 bp Hincll
fragment of pRep4. Overexpression of the fusion proteins is illustrated
in Figure 4 (B). In all cases, overexpression of a protein of the expected
molecular weight is apparent on SDS-PAGE of the crude lysate.
Purification of the coexpressed proteins under denaturing conditions
yielded two bands of apparent homogeneity upon analysis by Coomassie-
stained SDS-PAGE (Fig. 4B).
Stability Mutants
Applicants generated mutants of F[3] to test whether
reconstitution of mDHFR activity by fragment assembly was specific.
Protein stability can be reduced by changing the side-chain volume in the
hydrophobic core of a protein9, 22-25. Residue 11e114 of mDHFR occurs in
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a core b-strand at the interface between F[1,2] and F[3], isolated from the
active site. Ile 114 is in van der Waals contact with 11e51 and Leu93 in
F[1,2]11. We mutated Ile 114 to Val, Ala, or Gly. Figure 3 (panel B)
illustrates the results of cotransformation of E. coil with construct Z-F[1,2]
and the mutated Z-F[3] constructs. The colonies obtained from
cotransformation with Z-F[3:11e114Ala] grew more slowly than those
cotransformed with Z-F[3] or Z-F[3:11e114Val] (see inset to Fig. 3B). No
colony growth was detected in cells cotransformed with Z-F[3:11e114Gly].
The number of transformants obtained was not significantly different in
the case where colonies were observed, implying that cells cotransformed
with Z-F[1,2] and either Z-F[3], Z-F[3:11e114Val] or Z-F[3:11e114Ala] have
an equal survival rate. Overexpression of the mutants Z-F[3:11e114X] was
in the same range as Z-F[3], as determined by Coomassie-stained SDS-
PAGE (data not shown).
We also compared the relative efficiency of reassembly
of mDHFR fragments by measuring the doubling time of the
cotransformants in liquid medium. Doubling time in minimal medium was
constant for all transformants (data not shown). Selective pressure by
trimethoprim in the absence of 1PTG prevented growth of E. coli except
when transformed with pQE-16 coding for full-length DHFR due to low
levels of expression in uninduced cells. Induction of mDHFR fragment
expression with IPTG allowed survival of cotransformed cells (except in
the case of Z-F[1,2] + Z-F[3:11e114Gly], although the doubling times were
significantly increased relative to growth in the absence of trimethoprim.
The doubling time measured for cells expressing Z-F[1,2] + Z-F[3], Z-
F[1,2] + Z-F[3:11e114Val] and Z-F[1,2] + Z-F[3:11e114Ala] were 1.6-fold,
1.9-fold and 4.1-fold, higher respectively, than the doubling time of E. coil
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expressing pQE-16 in the absence of trimethoprim and IPTG. The
presence of IPTG unexpectedly prevented growth of E. coli transformed
with full-length mDHFR. Growth was partially restored by addition of the
folate metabolism end-products thymine, adenine, pantothenate, glycine
and methionine (data not shown). This suggests that induced
overexpression of mDHFR was lethal to E. coli when grown in minimal
medium as a result of depletion of the folate pool by binding to the
enzyme.
In another embodiment, applicants make point
mutations in the GCN4 leucine zipper of Z-F[1,2] and Z-F[3], for which
direct equilibrium and kinetic parameters are known and correlating these
known values with parameters derived from the PCA (Pelletier and
Michnick, in preparation). Comparison of cell growth rates in this model
system with rates for a DHFR PCA using unknowns would give an
estimate of the strength of the unknown interaction. This should enable
the determination of estimates of equilibrium and kinetic parameters for
a specific protein-protein interaction.
The present invention has illustrated and demonstrated
a protein-fragment complementation assay (PCA) based on mDHFR,
where a leucine zipper directs the reconstitution of DHFR activity. Activity
was detected by an E. coil survival assay which is both practical and
inexpensive. This system illustrates the use of mDHFR fragment
complementation in the detection of leucine zipper dimerization and could
be applied to the detection of unknown, specific protein-protein
interactions in vivo.
E. coil Aminoglycoside kinase: Optimization and Design of a PCA
using an Exonuclease-Molecular Evolution Strateay
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Although applicants have demonstrated that the
engineering/design strategy described above can be used to produce
complementary enzyme fragments, it is obvious that proteins did not
evolve in such a way that such fragments would be expected to have
optimal physical characteristics, including solubility, foldability (fast
folding), protease resistance, or enzymatic activity. An alternative
embodiment to the engineering/design strategy is the
endonuclease/evolution approach. This strategy can be used by itself or
in conjunction with the engineering/design strategy. The advantages of
this approach are that in principle, prior knowledge of the protein
strucuture is not necessary, that the optimal fragments are chosen for
PCA and that these fragments will also have optimal characteristics.
Following selection of optimal complementary fragments, the fragments
are exposed to multiple rounds of random mutagenesis. Mutagenesis is
acheived by suboptimal PCR combined with chemical mutagenesis or
DNA shuffling (Stemmer, W. P. C. (1994) Proc, Natl, Acad, Sci. USA 91,
10747-10751). The overall strategy is described for the case of
aminoglycoside kinase (AK), an example of antibiotic resistance marker
that can be used for dominant selection of prokaryotic cells such as E.
coli or eukaryotic cells such as yeast or mammalian cell lines. The
structure of an AK is already known, and so strategy (1) would be
possible, however we chose to combine both strategy (1) as defined for
DHFR above, in conjunction with strategy (2).
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EXPERIMENTAL PROTOCOL
The optimization/selection procedure is as follows:
= Generation of of library of AK fragments based on products of
Exonuclease digestion
Nested sets of deletions are created at the 5' and the 3'
ends of the AK gene. In order to create unidirectional deletions, unique
restriction sites are introduced in the regions flanking the AK gene. At the
5' and 3' termini, an "outer" sticky site with a protruding 3' terminus (Sph
I and Kpn I, respectively) and an "inner" sticky site with recessed 3'
terminus (Bgl II and Sal I, respectively) are added by PCR. Cleavage at
Sph I and Bgl II (or Kpn I and Sal I) results in creation of a protruding
terminus leading back to the flanking sequence and a recessed terminus
leading into the AK gene. Digestion with E. coli exonuclease III and S1
nuclease (Henikoff, S. (1987) Methods in Enzymology 155, 156-165)
yields a set of nested deletions from the recessed terminus only. Thus,
10 mg of DNA is digested with Sph I and Bgl ll (or Kpn I and Sal l),
phenol-chloroform extracted, and 12.5 U exonuclease III added. At 30
sec intervals over 10 min, aliquots are taken and put into solution with 2
U S1 nuclease. The newly created ends are filled in with T4 DNA
polymerase (0.1 U per sample) and the set of vectors closed back by
blunt-ended ligation (10 U ligase per sample). The average length of the
deletion at each time point is determined by restriction analysis of the
sets. This yields sets of AK genes deleted from the 5' or the 3' termini.
This manipulation is undertaken directly in the pQE-32-Zipper constructs,
such that the products can be used directly in activity screening.
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Screening for AK activity
As a first step in determining the requirements for
fragment complementation, we must determine the minimum N-terminal
and C-terminal fragments of AK that, alone, are active. Sets of deletions
are individually transformed into E. coli BL21 cells and expression of the
AK fragments is induced by IPTG. The sets where a significant number
of colonies appear in the presence of G418 serve to indicate the
approximate length of N- and C-terminal AK fragments which retain
activity. Fragment complementation must therefore be undertaken with
fragments taken from within these limits. The zipper-directed fragment
complementation is detected as follows: appropriate sets of deletions, or
pools of sets, are cotransformed into BL21, expression is induced with
IPTG and growth in the presence of varying G418 concentrations is
monitored. Large colonies which grow in the presence of high G418
concentrations are selected as giving the most efficiently complementing
products.
Directed evolution of optimal AK fragements using "DNA shuffling
After optimal fragments have been selected, the
individual fragments are removed by restriction digestion at Sph I and
Kpn I allowing for 5' and 3' constant priming regions flanking the N- or C-
terminal complementary fragments of AK. These oligonucleotides (2-4
mg) are digested with DNasel (0.005 units/ul, 100 ul) and fragments of
10-50 nucleotides are extracted from low melting point agarose. PCR is
then performed with the fragmented DNA, using Taq polymerase (2.5
units/up in a PCR mixture containing 0.2 mM dNTPs, 2.2 mM Mg2CI (or
0 mM for subuptimal PCR), 50 mM KCI, 10 mM Tris.HCI, pH 9.0, 0.1%
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TritonX-100.* A PCR program of 940/60 sec.; 940 30 sec.; 550 30 sec.;
720 30 sec. times 30 to 50; 720 5 mift Samples are taken every 5
cycles after 25 cycles to monitor the appearance of reassembled
complete fragments on agarose gel. The primeness PCR product is then
diluted 1:40 or 1:60 and used as template for PCR with 5', 3'
complementary constant region oligos as primers for a further 20 cycles.
Final product is restriction digested with Sph I and Kpn I and the products
subcloned back into pQE32-Zipper to yield the final library of expression
plasmids. As before, E. coil BL21 cells are sequentially transformed with
C-terminal or N-terminal complementary fragment-expression vectors at
an estimated efficiency of 109 and finally cells cotransformed with the
complementary fragment. E. coil are grown on agarose plates containing
1 mg/ ml G418 and after 16 hours the largest colonies are selected and
grown in liquid medium at increasing concentrations of G418. Those
clones showing the maximal resistance to G418 are then selected and if
maximum resistance or greater is reached the evolution is terminated.
Otherwise the DNA shuffling proceedure is repeated. Finally, optimal
fragments are sequenced and physical properties and enzymatic activity
are assessed. This optimized AK PCA is now ready to test for dominant
selection in any other cell type including yeast and mammalian cell lines.
This strategy can be used to develop any PCA based on enzymes that
impart dominant or recessive selection to a drug or toxin or to enzymes
that produce a colored or fluorescent product. In the later two cases the
end point of the evolution process is at minimum, reatainment of signal
for the intact, wild type enzyme or enhancement of the signal. This
strategy can also be used in the absense of knowledge of the enzyme
structure, whether the enzyme in mono-, di- or multimeric structure.
* trade mark
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However, knowledge of the enzyme structure does not preclude applying
this strategy as well, as described below.
As can be appreciated, knowledge of the enzyme
structure can be used to render a more efficient way of using molecular
evolution to design a PCA. In this case, the enzyme structure is used to
define minimal domains of the protein in question, as was done for DHFR.
Instead of generating fragments of completely random length for the N-
or C-terminal fragments, we select, during the exonuclease phase, those
fragments that at a minimum will code for one of the two domains. For
instance, in the case of AK, two well defined domains can be discerned
in the structure consisting of residues 1-94 in the N-terminus and residues
95-267 in the C-terminus. Endonuclease digestions are performed as
above, but reaction products are selected that will minimally code for one
of the two domains. These are then the starting points for fragment
selection and evolution cycles as described above.
Heteromeric Enzyme PCA
A further embodiment of the invention relates to PCA
based on using heterodimeric or heteromultimeric enzymes in which the
entire catalytic machinery is contained within one independently folding
subunit and the other subunit provides stability and/or a cofactor to the
enzymatic subunit. In this embodiment of PCA, the regulatory subunit is
split into complementary fragments and fused to interacting proteins.
These fragments are co-transformed/transfected into cells along with the
enzyme subunit. As with single enzyme PCA described for DHFR and
AK, reconstitution and detection of enzyme activity is dependment on
oligomerization domaiN-assisted reassembly of the regulatory subunit
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reassembly into its native topology. However, the reconstituted subunit
then interacts with the intact enzymatic subunit to produce activity. This
approach is reminiscent of the USPS system, except it has the advantage
that the enzyme in this case is not a constitutive cellular enzyme, but
rather an exogenous gene product. As such there is no problem with
background activity from the host cell, the enzyme can be expressed at
higher levels than a natural gene and can also be modified to be directed
to specific subcellular compartments (by subcloning compartment-specific
signal peptides onto the N- or C-termini of the enzyme and subunit
fragments). The specific advantage of this approach is that while the
single enzyme strategy may lead to suboptimal enzymatic activity, in this
approach, the enzyme folds independently and may in fact act as a
chaperone to the fragmented regulatory subunit, aiding in its refolding.
In addition, folding of the fragments may need not be complete in order
to impart regulation of the enzyme. This approach is realized by a
colorimetric/fluorometric assay we have developed based on the
Streptomyces tyrosinase. This enzyme catalyzes the conversion of
tyrosine to deoxyphenylalanine (DOPA). The reaction can be measured
by conversion of fluorocinyl-tyrosine to the DOPA form. The active
enzyme consists of two subunits, the catalytic domain (Melc2) and a
copper binding domain (Meld). Meld is a small protein of 14 kD that is
absolutely required for Melc2 activity. In the assay we are developing, the
Meld protein is split into two fragments that serve as the
complementation part of the PCA. These fragments, fused to
oligomerization domains, are coexpressed with Melc2, and the basis of
the assay is that Melc2 activity is dependent on complementation of the
Meld fragments. Stoichiometries of protein complexes can also be
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addressed (i.e. whether a complex consists of two or three proteins) as
follows. One fuses two proteins to the two Meld l fragments and a third to
intact Melc2. It thus can be shown that the minimum complementary
active complex of the tyrosinase will require that all three components
and therefore a trimer is necessary. A key aspect of this approach is that
we can easily demonstrate specific interactions by making one
component, specifically the protein-Melc2 fusions catalytic subunit
dependent on the other components by underexpressing it in the
background of overexpressed Meld 1 fragment-protein fusions.
Mu!timer Disruption-Based PCA
Although applicants have described only fragment complementation of
intact proteins, protein domains or subunits as comprising PCA, an
alternate enmbodiments relates to PCAs based on the disruption of the
interface between, for instance a dimeric enzyme that requires stable
association of the subunits for catalytic activity. In such cases, selective
or random mutagenesis at the subunit interface would disrupt the
interaction and the basis of the assay would be that oligomerization
domains fused to the subunits would provide the nessesary binding
energy to bring the subunits together into a functional enzyme.
Vector Design in Application to PCAs
The PCA strategies listed thus far have used two-
plasmid transformation strategies for expression of complementary
fragments. This approach has some advantages, such as using different
drug resistance markers to select for optimal incorporation of genes, for
instance in transformed or transfected cells or for optimum transformation
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of complementary plasmids into bacteria and control of expression levels
of PCA fragements using different promoters. However, single plasmid
strategies have advantages in terms of simplicity of transfection/
transformation. Protein expression levels can be controlled in different
ways, while drug selection can be achieved in one of two ways: In the
case of PCAs based on survival assay using enzymes that are drug
resistance markers themselves, such as AK, or where the enzyme
complements a metabolic pathway, such as DHFR, no additional drug
resistance genes need be incorporated in the expression plasmids. If
however the PCA is based on an enzyme that produces a colored or
fluorescent product, such as tyrosinase or firefly luciferase, an additional
drug resistance gene must be expressed from the plasmid. Expression
of PCA complementary fragments and fused cDNA libraries/target genes
can be assembled on single plasmids as individual operons under the
control of separate inducible or constitutive promotors, or can be
expressed polycistronically. In E. coil polycistronic expression can be
achieved using known intercoding region sequences, for instance we use
the region in the mel operon from which we derived the tyrosinase melci-
melc2 genes which we have shown to. be expressed at high levels in E.
coil under the control of a strong (tac) promoter. Genes could also be
expressed and induced off of independent promoters, such as tac and
arabinose. For mammalian expression systems, single plasmid systems
can be used for both transient or stable cell line expression and for
constitutive or inducible expression. Further, differential control of the
expression of one of the complementary fragment fusions, usually the
bait-fused fragment, can be controlled to minimize expression. This will
be important in reducing background non-specific interactions. Examples
_
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of differential control of complementary fragment expression include the
following strategies:
i) In polycistronic expression, transient or stable, expression of the
second gene will necessarily be less efficient and so this in itself could
serve to limit the quantity of one of the complementary fragments.
Alternatively, the first gene product can be limited in expression by
mutation of an upstream donor/splice site, while the second gene can be
put under the control of a retroviral internal initiation site, such as that
of
ECMV to enhance expression.
ii) Individual complementary fragment-fusion pairs can also be put under
the control of inducible promoters, all comercially available including
those based on Tet-responsive PhCMV*-1 promoter, and/or steroid
receptor response elements. In such a system the two complementary
fragment genes can be turned on and expression levels controlled by
dose dependent expression with the inducer, in these cases tetracycline
and steroid hormones.
EXAMPLE 2
Applications of the PCA strateay to detect novel gene products in
biochemical Pathways and to map such pathways
Among the greatest advantage of PCA over other
molecular interaction screening methods is that they are designed to be
performed both in vivo and in any type of cell. This feature is crucial if the
goal of applying a technique is to identify novel interactions from libraries
and simultaneously be able to determine if the interactions observed are
biologically relevant. The detailed example given below, and other
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examples at the end of this section illustrate how it is that validation of
interactions with PCA is possible. In essence, this is achieved as follows.
In biochemical pathways, such as hormone receptor-mediated signaling,
a cascade Of enzyme-mediated chemical reactions are triggered by some
molecular event, such as by hormone binding to its membrane surface
receptor. Enzyme interactions with protein substrates and protein-protein
or protein-nucleic acid interactions with enzyme-modified substrates then
occur. Such biochemical signaling cascades only occur in specific cell
types and model cell lines for studying these processes. Therefore, to
detect induced interactions, such as with known proteins in a pathway
with yet unidentified proteins, one obviously needs to perform such
screening in appropriate model cell lines and in the correct cellular
compartment. Only the PCA strategy can be used in a general way to do
this. Protein-molecular interaction techniques such as yeast two- or
three-hybrid techniques cannot be performed in a context where such
events occur, except in the limiting case of nuclear interaction in yeast or
interactions that are not triggered. There do exist mammalian two-hybrid
techniques where it might be possible to detect induced protein
interactions, but only again if the proteins involved can be simultaneously
activated, transported to the nucleus and interact with their partners.
PCAs do not have these limitation since they do not require additional
cellular machinery available only in specific compartments. A further
point is that by performing the PCA strategy in appropriate model cell
types, it is also possible to introduce appropriate positive and negative
controls for studying a particular pathway. For instance, for a hormone
signaling pathway it is likely that hormone signaling agonists and
antagonists or dominant-negative mutants of signaling cascade proteins
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would be known, that are upstream or act in parallel to the events being
examined in the PCA. These reagents could be used to determine if
novel interactions detected by the PCA are biologically relevant. In
general then, interactions that are detected only if hormone is introduced
but are not seen if an antagonist is simultaneously introduced could be
hypothesized to represent interactions relevant to the process under
study.
Below is a detailed description of an application of the
DHFR that illustrates these points, as well as further examples where the
PCA strategy could be used.
Application of the DHFR PCA to Mapping Growth Factor-Mediated
Signal Transduction Pathways
One of the earliest detectable events in growth factor-
activated cell proliferation is the serine phosphorylation of the S6 protein
of the 40S ribosomal subunit. The discovery of serine/threonine kinases
that specifically phosphorylate S6 have considerably aided in identifying
novel mitogen mediated signal transduction pathways. The
serine/threonine kinase p70S6k has been identified as a specific S6
phosphorylase131136. p70S6k is activated by serine and threonine
phosphorylation at specific sites in response to several mitogenic signals
including serum in serum starved cells, growth factors including insulin
and PDGF, and by mitogens such as phorbol esters. Considerable effort
has been made over the last five years to determine how p70/p85S6k are
activated in response to mitogens. Two receptor-mediated pathways have
been implicated in p70S6k activation, one associated with the
phosphatidylinosito1-3-kinase (PI(3)k) and the other with the P1(3)k
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homologue rnTOR137-144. Key to understanding of this proposal, is the fact
that the role of these enzymes in activation of p70S6k was determined by
effects of two natural products on phosphorylation and enzyme activity:
rapamycin, which indirectly inhibits mTOR activity, and wortmannin, which
directly inhibits P1(3)k activity. It is also important to note that no direct
upstream kinases or other regulatory proteins of p70S6k have been
identified to this date.
The interactions of p70S6k with its known substrate 86
can be studied as a test system for the DHFR PCA in E. coli and in
mammalian cell lines. One can also seek to identify novel interactions
with this enzyme that would lead to new insights into how this important
enzyme is regulated. Also, since activation of the enzyme is mediated by
multiple pathways that can be selectively inhibited with specific drugs, this
is an ideal system to test PCAs as methods to distinguish induced versus
constitutive protein-protein interactions.
a) Testing of the E. con survival assay: Interaction of p70S6k with S6
This test is ideal, because the apparent Km (= 250 nM)
of p7086k for S6 protein145 is approximately the same as the Kd for
leucine zipper-forming peptides from GCN4 used in our test system.
However, we will have to use a constitutively active form of the enzyme
for our tests. An N-terminal truncated form of the enzyme D77-p70S6k,
is constitutively active and will be used in these studies147.
Methodology: D77-p70S6k-F[1,2] fusion and D77-p70S6k-F[3] fusion, or
F[1,2] and D77-p70S6k-F[3] fusion (as a control) will be cotransformed
into E. coil and the cells grown in minimal medium in the presence of
trimethoprim. Colonies will be selected and expanded for analysis of
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kinase activity against 40S ribosomal subunits, and for coexpression of
the two proteins.
b) Modification of the bacterial survival assay for library screening:
Identification of Novel Interacting Proteins
Screening an expression library for interactions with a
given target (p70S6k-D77, in this case) will be straightforward in this
system, given that the only steps involved are: 1-construction of the
fusion-expression library as a fusion with mDHFR fragment[3]; 2-
transformation of the library in E. coli BL21 harboring pRep4 (for
constitutive expression of the lac repressor; this is required in the case
where a protein product is toxic to the cells) and a plasmid coding for the
fusion: p70S6k-D7741 ,2]; 3-plating on minimal medium in the presence
of trimethoprim and 1PTG; 4-selection of any colonies that grow,
propagation and isolation of plasmid DNA, followed by sequencing of
DNA inserts; 5-purification of unknown fusion products via the hexaHis-
tag and sizing on SDS-PAGE.
Methodoloay:
The overall strategy is illustrated in Figure 5. 1-
Construction of a directional fusioN-expression library: i-cDNA production:
One can isolate poly(A)+ RNA from BA/F3 cells (B-lymphoid cells)
because these cells have successfully been used in the study of the
rapamycin-sensitive p70S6k activation cascade139. To enrich for full-
length mRNA, we will affinity purify the mRNA via the 5' cap structure by
the CAPture method'. Reverse transcription will be primed by a "Linker
Primer": it has a poly(T) tail to prime from the poly(A) mRNA tail, and an
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Xhol site for later use in directional subcloning of the fragments. The first
strand is then methylated. After second strand synthesis and blunting of
the products, "EcoRI Adapters" are added, pruducing digestion of the
linkers with EcoRI and Xhol (the inserts are protected by methylation)
produces full-length cDNA ready for directional insertion in a vector
opened with EcoRI and Xhol. Because the success of library screening
depends largely on the quality of the cDNA produced, we will use the
above methods as they have proven to consistently produce high-quality
cDNA libraries. ii-Insertion of /he cDNA into vectors: The library will be
constructed as a C-terminal fusion to mDHFR F[3] in vector pQE-32
(Qiagen), as we have obtained high levels of expression of mDHFR
fusions from this vector in BL21 cells. Three such vectors will be created,
differing at their 3' end, which is the novel polycloning site that we
engineered (described earlier, under Methods), carrying either 0, 1, or 2
additional nucleotides. This allows read-through from F[3] into the library
fragments in all 3 translational reading frames. The cDNA fragments will
be directionally inserted at the EcoRI and Xhol sites in all three vectors
at once. 2, 3, 4, and 5- These steps have been described earlier, under
Results, apart from the final sequencing of clones identified using
sequencing primers specific to vector sequences flanking sites of library
insertion. The protein purification will also be as described earlier, by a
one-step purification on Ni-NTA (Qiagen). If the product size is more than
15 kDa over the molecular weight of the DHFR component (equal to a
cDNA insert of more than 450bp), we will have the inserts sequenced at
the Sheldon Biotechnology Center (McGill University).
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c) Development of the Eukaryotic Assay
The transformation of the system described above, is
useful to produce an equivalent assay for use in eukaryotic cells. The
basic principle of the assay is the same: the fragments of mDHFR are
fused to associating domains, and domain association is detected by
reconstitution of DHFR activity in eukaryotic cells (Figure 5).
Creation of the expression constructs: The DNA fragments coding for
the GCN4-zipper-mDHFR fragment fusions were inserted as one piece
into pMT3, a eukaryotic transient expression vector126. Expression of the
fusion proteins in COS cells was apparent on SDS-PAGE after 35[S]Met
labeling.
Survival assays in eukaryotic cells: Two systems can be used for
detection of mDHFR reassembly, in parallel: i- CHO-DUKX B11 cells
(Chinese Hamster Ovary cell line deficient in DHFR activity) are
cotransfected with GCN4-zipper-mDHFR fragment fusions. The cells are
grown in the absence of nucleotides; only cells carrying reconstituted
DHFR will undergo normal cell division and colony formation. ii-
Methotrexate (MTX)-resistant mutants of mDHFR have been created, with
the goal of transfecting cells that have constitutive DHFR activity such as
COS and 293 cells. We mutated F[1,2] in order to incorporate, one at a
time, each of five mutations that significantly increase Ki (MTX):
Gly15Trp, Leu22Phe, Leu22Arg, Phe31Ser and Phe34Ser (numbering
according to the wild-type mDHFR sequence). These mutations occur at
varying positions relative to the active site and relative to F[3], and have
varying effects on Km (DHF), Km (NADPH) and Vmax of the full-length
mammalian enzymes in which they were. Mutants Z-F[1,2: Leu22Phe],
Z-F[1,2: Leu22Arg] and Z-F[1,2: Phe31Ser] all allowed for bacterial
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survival with high growth rates when cotransformed with Z-F[3] (results
not shown).The five mutants will be tested in eukaryotic cells, in
reconstitution of mDHFR fragments to produce enzyme that can sustain
COS or 293 cell growth while under the selective pressure of MTX, which
will eliminate background due to activity of the native enzyme. The
mutations offers an advantage in selection while presenting no apparent
disadvantage with respect to reassembly of active enzyme. If the
reconstituted mDHFR produced in either of the survival assays allows
eukaryotic cell growth that is significantly slower than growth with the wild-
type enzyme, thymidylate will be added to the growth medium to partially
relieve the selective pressure offered by the lack of nucleotides.
d) Testing of the eukaryotic survival assay
It is necessary at the outset to test whether induced
interactions with p70S6k can be detected. One can use the same test
system as that for the E. coli test system described above: Induction of
association of p70S6k with S6 protein.
Methodology:
mDHFR Leu22Phe mutant S6-F[1,2] and p70S6k-F[3], or F[1,2] and
p70S6k-F[3] (as a control) will be cotransfected into COS cells and the
cells will be serum starved for 48 hours followed by replating of cells at
low density in serum and MTX. Colonies will be selected and expanded
for analysis of kinase activity against 40S ribosomal subunits, and for
coexpression of the two proteins. Further controls will be performed for
inhibition of protein association with wortmannin and rapamycin.
e) Modification of the eukaryotic survival assay for library screening
An important part of the work required in creating a
library for use in eukaryotic cells will have been accomplished already, as
_ _
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the EcoRi/Xhol directional cDNA produced by the Stratagene "cDNA
Synthesis Kit" can directly be inserted directionally into the Stratagene
Zap Express vector.
Methodology:
Steps 1 through 5 are parallel to those for the bacterial
library screening (above). 1-Again, the library is constructed as a C-
terminal fusion to mDHFR F[3]. F[3] (with no stop codon) will be inserted
in frame in Zap Express, followed by insertion of the novel polylinkers
allowing expression of the inserts in all three reading frames (described
above), and by the EcoRI/Xhol directional cDNA. This bacteriophage
library will be propagated and treated with the Stratagene helper phage
to excise a eukaryotic expression phagemid vector (pBK-CMV) carrying
the fusion inserts. 2-Cotransfection of the library and p70S6k-F[1,2]
constructs in eukaryotic cells: we will perform the screening in COS or
293 cells, as these are responsive to serum in activating the p70S6k
signaling pathway. Selection experiments will be performed as described
for the S6 test system above. 3-Propagation, isolation and sequencing
of the insert DNA will be undertaken. 4-The cloned fusion proteins will be
sized on SDS-PAGE by direct visualization after 35S-Met/Cys labeling,
or by Western blotting using a commercial polyclonal antibody to mDHFR.
Generalization of the Strategy: The scheme for detecting partners for
the protein p70S6k can be applied to studies of any biochemical pathway
in any living organism. Such pathways may also be related to disease
processes. The disease-related pathway may be an intrinsic process of
cells in humans where a pathology arises from, for instance mutation,
deletion or under or over expression of a gene. Alternatively the
biochemical pathway may be one that is specific to a pathogenic
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organism or the mechanism of host invasion. In this case, component
proteins of such processes may be targets of a therapeutic strategy, such
as development of drugs that inhibit invasion by the organism or a
component enzyme in a biochemical pathway specific to the pathogenic
organism.
Inflamatory diseases are a case in point that can
concern both examples. The protein-protein interactions that mediate the
adhesion of leukocytes to inflamed tissues are known to involve such
proteins as vascular cell adhesion molecule-1 (VCAM-1), and certain
cytokines such as IL-6 and IL-8 that are produced during inflammation.
However, many of the proteins involved in onset of inflammatory
response remain unknown; further, the intracellular signaling pathways
triggered by the extracellular associations are poorly understood. The
PCAs could be used in elucidation of the mechanisms underlying the
onset of inflammation, as well the ensuing signaling. For example,
signaling pathways associated with inflamation, such as those mediated
by IL-1, IL-6, IL-8 and tumor necrosis have been studied in some detail
and many direct and downstream regulators are known. These
regulators can be used as starting point targets in a PCA screening to
identify other signalling or modulating proteins that could also be targets
for drug development.
There is an increased risk of infection by enteric
pathogens in the occurrence of the intestinal inflammation that
characterizes idiopathic intestinal diseases. There are two mechanisms
which need to be better understood here and which can be addressed by
PGA:
i- the cellular mechanisms of inflammation as described above, and
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ii- the discovery of the specific cell-surface ligands which the pathogenic
organisms recognize and associate with. Secreted proteins produced by
the pathogen can bind to the basolateral membrane of epithelial cells (as
in the case in Yersinia pseudotuberculosis infection) or be translocated
into intestinal epithelial cells (Salmonella infection), promoting infectivity
and/or physiological responses to the infection. However, in most cases
the interactions between the pathogenic protein and the epithelial cells
are unknown.
Cell adhesion and nervous system regeneration A related example in
cell adhesion includes processes involved in develoment and
regeneration in the nervous system. Cadherens are membrane proteins
that mediates calcium dependent cell-cell adhesion. To do so they need
another class of cytoplasmic proteins called cathenins. Those make a
bridge between cadherins and cytoskeleton. Cathenins are also regulate
genes that control differentiation-specific genes. For instance, the protein
B-cathenin can interact in certain situation with a transcription factor (lef-
1) and be translocated into the nucleus where it constrains the number of
genes transactivated by lef-1 (differentiation). This process is regulated
by the Wnt signaling pathway (homologs to the wingless pathway in
drosophila) by inactivation of GSK3B which permit degradation after of
APC (a cytoplasmic adapter protein). PCA strategies could be used to
identify novel proteins involved in the regulation of these processes.
Proteins involved in viral integration processes are
examples of targets that could be tested for inhibitors using the PCA
strategies. Examples for the HIV virus include:
1
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i) inhibition of integrase or the transport of the pre-integration complex:
protein Ma or vpr.
ii) Inhibition of the cell cycle in G2 by vpr (interaction by cyclin B)
causing
induction of apoptosis.
iii) Inhibition of the interaction of gp160 (precursor of the membrane
proteins) with furine.
Accessory proteins of HIV as a therapeutic target:
i) Vpr: nuclear localizing sequence (target): interaction site of vpr with
phosphatasesA .
ii) vif: interaction with vimentin (cytoskeleton associated protein).
ii) Vpu: Degradation of CD4 in the RE mediated by the cytoplasmic tail of
Vpu.
iii) nef: Myristoylation signal of Nef.
EXAMPLE 3
Other Examples of Protein Fragment Complementation Assays
Other examples of assays are herein examplified. The
reason to produce these assays is to provide alternative PCA strategies
that would be appropriate for specific protein association problems such
as studying equilibrium or kinetic aspects of assembly. Also, it is possible
that in certain contexts (for example, specific cell types) or for certain
applications, a specific PCA will not work but an alternative one will.
Further below are brief descriptions of each other PCAs embodiments.
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1) Glutathione-S-Transferase (GST) GST from the flat worm
Schistosoma japonicum is a small (28 kD), monomeric, soluble protein
that can be expressed in both prokaryotic and eukaryotic cells. A high
resolution crystal structure has been solved and serves as a starting point
for design of a PCA. A simple and inexpensive colorimetric assay for
GST activity has been developed consisting of the reductive conjugation
of reduced glutathione with 1-chloro-2,4-dinitrobenzine (CNDB), a brilliant
yellow product. We have designed a PCA based on similar structural
criteria used to develop the DHFR PCA using GCN4 leucine zippers as
oligomerization domains. Cotransformants of zipper-GST-fragment
fusions are expressed in E. coil on agar plates and colonies are
transferred to nitrocellulose paper. Detection of fragment
complementation is detected in an assay where a glutathione-CDNB
reaction mixture is applied as an aerosol on the nitrocellulose and
colonies expressing co-expressed fragments of GST are detected as
yellow images.
2) Green Fluorescent Protein (GFP) GFP from Aequorea victoria is
becoming one of the most popular protein markers for gene expression.
This is because the small, monomeric 238 amino-acids protein is
intrinsically fluorescent due to the presence of an internal chromophore
that results from the autocatalytic cyclization of the polypeptide backbone
between residues Ser65 and Gly67 and oxidation of the - bond of Tyr66.
The GFP chromophore absorbs light optimally at 395 nm and possesses
also a second absorption maximum at 470 nm. This bi-specific absorption
suggests the existence of two low energy conformers of the chromophore
whose relative population depends on local environment of the
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chromophore. A mutant Ser65Thr that eliminates isomerization (single
absorption maximum at 488 nm) results in a 4 to 6 times more intense
fluorescence than the wild type. Recently the structure of GFP has been
solved by two groups, making it now a candidate for a strucutre-based
PCA-design, which we have begun to develop. As with the GST assay,
we are doing all of our initial development in E. coli with GCN4 leucine
zipper-forming sequences as oligomerization domains. Direct detection
of fluorescence by visual observation under broad spectrum UV light will
be used. We will also test this system in COS cells, selecting for co-
transfectants using fluorescence activated cell sorting (FAGS).
3) Fire Fly Luciferase. Firefly luciferase is a 62 kDa protein which
catalyzes oxidation of the heterocycle luciferin. The product posesses
one of the highest quantum yields for bioluminescent reactions: one
photon is emitted for every oxidized luciferin molecule. The structure of
luciferase has recently been solved, allowing for strucutre-based
development of a PCA. As with our GST assay, cells are grown on a
nitrocellulose matrix. The addition of the luciferin at the surface of the
nitrocellulose permits it to diffuse across the cytoplasmic membranes and
trigger the photoluminescent reaction. The detection is done immediately
on a photographic film. Luciferase is an ideal candidate for a PCA: the
detection assays are rapid, inexpensive, very sensitive, and utilizes non-
radioactive substrate that is available commercially. The substrate of
luciferase, luciferin, can diffuse across the cytoplasmic membrane (under
acidic pH), allowing the detection of luciferase in intact cells. This
enzyme is currently utilized as a reporter gene in a variety of expression
systems. The expression of this protein has been well characterized in
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bacterial, mammalian, and in plant cells, suggesting that it would provide
a versatile PCA.
4) Xanthine-guanine phosphoribosyl transferase (XGPRT) The E. coil
enzyme XGPRT converts xanthine to xanthine monophosphate (XMP),
a precursor of GMP. Because the mammalian enzyme hypoxanthine-
guanine phosphoribosyl transferase HGPRT can only use hypoxanthine
and guanine as substrates, the bacterial XGPRT can be used as a
dominant selection assay for a PCA for cells grown in the presense of
xanthine. Vectors expressing XGPRT confer the ability of mammalian
cells to grow in selective medium containing adenine, xanthine, and
mycophenolic acid. The function of mycophenolic acid is to inhibit de
novo synthesis of GMP by blocking the conversion of IMP into XMP
(Chapman A. B., (1983) Molec. & Cellul. Biol. 3, 1421-1429). The only
GMP produced then come from the conversion of xanthine into XMP,
catalyzed by the bacterial XGPRT. As with aminoglycoside
phosphotransferase fragments of XGPRT can be generated based on the
known structure (See table 1.) using the design-evolution strategy
described above with fragments fused to the GCN4 leucine zippers as a
test oligomerization domains. The complementary fusions are
cotransfected and the proteins transiently expressed in COS-7 cells, or
stability expressed in CHO cells, grown in the selective medium. In the
case of CHO cells, colonies are collected and sequentially re-cultured at
increasing concentrations of the selective compounds in order to enrich
for populations of cells that efficiently express the fusions at high
concentrations.
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5) Adenosine deaminase Adenosine deaminase (ADA) is present in
minute quantities in virtually all mammalian cell. Although it is not an
essential enzyme for cell growth, ADA can be used in a dominant
selection assay. It is possible to establish growth conditions in which the
cells require ADA to survive. ADA catalyzes the irreversible conversion
of cytotoxic adenine nucleosides to their respective nontoxic inosine
analogues. By adding cytotoxic concentrations of adenosine or cytotoxic
adenosine analogues such as 9-b-D-xylofuranosyladenine to the cells,
ADA is required for cell growth to detoxify the cytotoxic agent. Cells that
incorporate the ADA gene can then be selected for amplification in the
presence of low concentrations of 20-deoxycoformycin, a tight-binding
transition state analogue inhibitor of ADA. ADA can then be used for a
PCA based on cell survival (Kaufman, R. J. et al. (1986) Proc. of the Nat.
Acad. Sci. (USA) 83, 3136-3140). As with the other systems described
above, fragments of ADA can be generated based on the known structure
(See table 1.) using the design-evolution strategy described above with
fragments fused to the GCN4 leucine zippers as a test oligomerization
domains. The complementary fusions are cotransfected and the proteins
transiently expressed in COS-7 cells, or stability expressed in CHO cells,
grown in the selective medium containing 20-deoxycoformycin. In the
case of CHO cells, colonies are collected and sequentially re-cultured at
increasing concentrations of 20-deoxycoformycin in order to enrich for
populations of cells that efficiently express the fusions at high
concentrations
6) Bleomycin binding protein (zeocin resistance gene) Zeocin, a
member of the bleomycin/phleomycin family of antibiotics, is toxic to
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bacteria, fungi, plants, and mammalian cells. The expression of the
zeocin resistance gene confers resistance to bleomycin/zeocin. The
protein confers resistance by binding to and sequestering the drug and
thus preventing its association and hydrolysis of DNA. Berdy, J. (1980)
In Amino Acid and Peptide Antibiotics, J. Berdy, ed. (Boca Raton, FL:
CRC Press), pp.459-497, Mu!sant, P., Tiraby, G., Kallerhoff, J., and
Perret, J. (1989 Somat. Cell. Mol. Genet. 14, 243-252). Bleomycin binding
protein (BBP) could then be used for a PCA based on cell survival. As
with the other systems described above, fragments of ADA can be
generated based on the known structure (See table 1.) using the design-
evolution strategy described above with fragments fused to the GCN4
leucine zippers as a test oligomerization domains. The BBP is a small (8
kD) dimer that binds to drugs via a subunit interface binding site. For this
reason, the design would be somewhat different in that first, a single
chain form of the dimer would be generated by making a fusion of two
BBP genes with a short sequence coding for a simple polypeptide linker
introduced between the two subunits. Fragments in this case will be
based on a short sequence of one of the subunit modules, while the other
fragment will be composed of the remaining sequence of the subunit plus
the other subunit. Complementation and selection experiments will be
performed as described for the examples above using bleomycin or
zeocin as selective drugs.
7) Hygromycin-B-phosphotransferase The antibiotic hygromycin-B is
an aminocyclitol that inhibits protein synthesis by disrupting translocation
and promoting misreading. The E. coli enzyme hygromycin-B-
phosphotransferase detoxifies the cells by phosphorylating Hygromycin-
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. B. When expressed in mammalian cells, hygromycin-B-
phosphotransferase can confer resistance to hygromycin-B ( Gritz, L.,
and Davies, J. (1983) Gene 25, 179-188.). The enzyme is a dominant
selectable marker and could be used for a PCA based on cell survival.
While the structure of the enzyme is not known it is suspected that this
enzyme is homologous to aminoglycoside kinase (Shaw, et at. (1993)
Microbiol. Rev. 57, 138-163). It is therefore possible to use the combined
design/evolution strategy to produce a PCA with this enzyme and perform
dominant selection in mammalian cells with selection at increasing
concentrations of hygromycin B.
8) L-histidinol NAD+oxydoreductase The hisD gene of Salmonella
typhimurium codes for the L-histidinol NAD+oxydoreductase that
converts histidinol to histidine. Mammalian cells grown in media lacking
histidine but containing histidinol can be selected for expression of hisD
(Hartman, S. C., R. C. Mulligan (1988) Proc. of the Nat. Acad. Sci. (USA)
85, 8047-8051). An additional advantage of using hisD in dominant
selection is that histidinol is itself toxic, inhibiting the activity of
endogenous histidyl-tRNA synthetase.. Histidinol is also inexpensive and
readily permeates cells. The structure of histidinol NAD+oxydoreductase
is unknown and so development of a PCA based on this enzyme is based
entirely on the exonuclease fragment/evolution strategy.
The following Table list alternative embodiments using other PCA
reporters. Abreviations in Table: Type: D, dominant selection marker; R,
recessive selection marker. Structure: four letter codes= Protein Data
Bank (PDB) entries; K, known but not deposited in PDB; U, unknown.
mono/oligo: M, monomer; D, dimer; tetra, tetramer.
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TABLE 1. A list of Other Potential PCA Reporter Candidates
A-Assays based on Dominant or Recessive Selection
mono/ Selection
Enzyme Type Structure Size oligo drugs/Conditions
DHFR R/D many 18kD M
methotrexate/trimetho
prim
Adenosine deaminase D/R 1ADD M Xyl-A or
adenosine,alanosine,
and 2'-
deoxycoformycin
Thymidine kinase D/R 1KI N D gangcyclovene, HAT
Mutant hypoxanthine- guanine D 1HGM D HAT + thymidine
phosphoribosyl transferase kinase
Thymidylate synthetase R 1NJE 35kd M 2
fluorodeoxyuridine
Xanthine-guanine D 1NUL mycophenolic acid
phosphoribosyl transferase with limiting
xanthine
Glutamine synthetase R 2LGS
Asparagine synthetase R U B-aspartyl
hydroxamate or
albizin
Puromycin N- D U 23kD M puromycin
acetyltransferase
Aminoglycoside D K 35kD M neomycin, G418,
phosphotransferase gentamycin
Hygromycin B D U M hygromycin B
phosphotransferase
L-histidinol:NAD+ D U 46kD M histidinol
oxidoreductase
Bleomycin binding protein D K 8kD D bleomycin/zeocin
Cytosine methyl-transferase R/D U 5-Azacytidine (5-
aza-
CR) and 5-aza-2'-
deoxycytidine
06-alkylguanine D 1ADN N-methyl-N-
alkyltransferase nitrosourea
Glycinamide ribonucleotide R 1GRC 23.2 D
dideazatetrahydrofolat
transformylase kD e, minus purine
Glycinamide ribonucleotide R U 45.9 minus purine
synthetase kD
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Enzyme Type Structure Size mono/ Selection
oligo drugs/Conditions
Phosphoribosyl- R U 36.7 minus purine
aminoimidazole synthetase kD
Formylglycinamide ribotide R U 141. M L-azaserine, 6-diazo-
amidotransferase 4kD 5-oxo-L-norleucine,
minus purine
Phosphoribosyl- R U 39.5 D minus purine
aminoimidazole carboxylase kD
"Phosphoribosyl- R U 57.3 minus purine
aminoimidazole carboxamide kD
formyltransferase
Fatty acid synthase R 272k D ceruienin
IMP dehydrogenase R 1AK5 55.4 Tetra mycophenolic acid
kD
ii-Viral Plaque Assays
Mono/ Selection
Enzyme Type Structure Size
Oligo drugs/Conditions
Thioredoxin D 1TDF 34.5kD D
Reverse transcriptase D 3HVT
Viral protease
B-Cell Death Assays
Enzyme Type Structure Size Mono/ Selection
Oligo drugs/Conditions
Cysteine protease: papain D 1STF 38.9kD M , inhibited by cystatin
Cysteine protease: D 1CP3 17kD + Hetero inhibited by DEVD-
aldehyde
caspase 12kD D (can also by used in a
fluorimetric or colorimetric
assay, in vitro)
Metalloprotease: D 47.1kD M inhibited by methyl-
ethyl
carboxypeptidase succinic acid
Serine protease: D 1PTK 30.6kD M inhibited by serpins
proteinase K
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Mono/ Selection
Enzyme Type Structure Size
Oligo drugs/Conditions
Aspartic protease: pepsin D 1PSN 34.5kD M inhibited by pepstatin
A (can
also be used in an
fluorimetric assay, in vitro)
Lysozyme D many 23.2kD M inhibited by N-
acetylglucosamine
trisaccharide
RNAse D many 13.3kD M inhibited by RNAse
inhibitor
DNAse D 1DNK 61.6kD M inhibited by actin
Phospholipase A2 D 1P2P 13.8kD M/D many inhibitors:
bromophenacyl bromide,
hexadecyl-trifluoroethyl-
giycero-phosphomethanol,
bromoenol lactone, etc.
Phospholipase C D 1AH7 28kD M many inhibitors:
neomycin,
chelerythrine, U73122, etc.
C-Colorimetric/Fluorimetric Assay
Mono/
Enzyme Structure Size Oligo Selection drugs/Conditions
DT-Diaphorase (NAD(P)H- 1QRD 26kD D NADPH-diaphorase stain,
[quinone acceptor) inhibited by dicumarol,
oxidoreductase) Cibacron blue and
phenidione
Note: can also be used in a cell
death assay
(+nitrobenzimidazole, fo
example).
(NAD(P)H-[quinone isoform of 21kD ' D NRH-diaphorase stain,
acceptor] oxidoreductase)-2 laRD inhibited by
pentahydroxyflavone
Thermophilic diaphorase 30kD M NADH-diaphorase stain
(Bacillus
stearothermophilus)
Glutathione-S-transferase 1GNE 26kD D production of a yellow
product
other by the conjugation of
isoform glutathione with an aromatic
of 28kD substance, chloro
dinitrobenzene (CDNB)
Luciferase 1LCI 62kD M Fluorometric
Green-fluorescent protein 1EMA 30kD M Intrinsic fluorescence
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Enzyme Structure Size Mono/ Selection drugs/Conditions
Oligo
Chloramphenicol 1CLA 25kD Tri Fluorimetric: Bodipy
acetyltransferase chloramphenicol
Uricase ,32kD Tetra Fluorometric
SEAP (secreted form of 1AJA M CSPD chemiluminescent
human placental alkaline substrate
phosphatase)
B-Glucuronidase 1BHG 71kD Tetra Histochemical, fluorometric or
spectrophotometric assays
using various substrates such
as X-GLUC.
D-Heteromeric Enzyme Strategies
Tyrosinase 30kD + Hetero Colorimetric: synthesis of
14kD M+M melanin
EXAMPLE 4
Examples of Variants of PCA to detect multiple protein/protein-
dna/protein RNA/protein-drug complexes
To this point specific examples have only been made of
applications of PCA to protein-pair interactions. However, it is possible
to apply PCA to multiprotein, protein-RNA, 'protein-DNA or protein-small
molecule interactions. There are two general schemes for achieving such
systems. Multi-subunit PCA: Two proteins need not interact for a PCA
signal to be observed; if a partner protein or protein complex binds to two
proteins simultaneously, it is possible to detect such a three protein
complex. A multusub unit PCA is conceived with the example of herpes
simplex virus thymidine kinase (TK), a homodimer of 40 kD . In this
conception, the TK structure contains two well defined domains consisting
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of an alpha/beta (residues 1-223) and an alpha-helical domain (224-374).
As a test system, we use the Rop1 dimer, a four helix bundle homodimer.
The two fragments of TK are extracted by PCR and subcloned into the
transient transfection vector pMT3, the first in tandem to the Adenovisus
major late promoter, tripartite leader 3' to the first ATG, and the second
downstream of a ECMV internal initiation site. Restriction sites previously
introduced between the first and the last ATG are subcloned into BamHI/
Kpnl and Pstl/EcoRI cloning sites downstream of the two ATGs. These
are used to subclone PCR-generated fragments of the Rop1 subunits into
two different vectors. Subsequently Ltk- cells are cotransfected by
lipofection with the two plasmids and colonies of surviving cells are
serially selected in medium containing increasing concentrations of HAT
(hypoxanthine/ aminopterin/thymidine).
Cells that express
complementary fragments of TK fused to the four Rop1 will proliferate
under this selective pressure, or otherwise die. Specific examples of use
of this concept would be in determining constituents of multiprotein
complexes that are formed transiently or constitutively in cells.The utility
of PCA is not limited to detecting protein-
protein interactions, but can be adapted to detecting interactions of
proteins with DNA, RNA, or small molecules. In this conception, two
proteins are fused to PCA complementary fragments, but the two proteins
do not interact with each other. The interaction must be triggered by a
third entity, which can be any molecule that will simultaneously bind to the
two proteins or induce an interaction between the two proteins by causing
a conformational change in one or both of the partners. Two examples
have been demonstrated in our lab using the mDHFR PCA in E. coil. In
the first case a natural product, the immunosuppressant drug rapamycin,
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is used to induce an interaction between its receptor FKBP12 and a
partner protein mTOR ( mammalian Target of Rapamycin). We detect
this by cotransformation of DHFR fragments fused to FKBP or mTOR into
E. coli grown in the presence or absence of trimethoprim (as described
above) and rapamycin (0- 10 nM). We have demonstrated that support
of growth as detected by colony formation is completely dependent on the
addition of rapamycin, suggesting that the mDHFR PCA is detecting a
rapamycin-induced assembly of a FKBP12-mTOR and subsequent
reconstitution of DHFR activity. This is one example of a use of the PCA
strategy to test for small molecules that can induce interactions between
proteins. General applications could be made to therapeutic
development, in the form screening small molecule combinatorial
compound libraries for molecules that induce interactions between
proteins, that may inhibit the activities of either or both of the proteins,
or
activate specific cellular processes that are initiated by other events, such
as growth factor-mediated receptor dimerization. The discovery of such
small molecules could lead to the development of orally available drugs
for the treatment of a broad spectrum of human diseases.
Another example of an induced interaction we have
studied with the DHFR PCA is the interaction of the oncogene GTPase
p21 ras and its direct downstream target, the serine/threonine kinase raf.
This interaction only occurs when the GTPase is in the GTP-bound form,
whereas turnover of GTP to GDP leads to release of the complex. As
with the FKBP-mTOR complex, we have demonstrated this induced
interaction in E. coll. PCA could be used in a general way to study such
induced interactions, and to screen for compounds that release or
prevent these interactions in pathological states. The ras-raf interaction
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itself could be a target of therapeutic intervention. Oncogenic forms of
ras consist of mutants that are incapable of turning over GTP and
therefore remain continuously associated with activated ras. This leads
to a constitutive uncontrolled growth signal that results, in part, in
oncogenesis. The identification of compounds that inhibit this process,
by PCA, would be of value in broad treatment of cancers. Other
examples of multimolecular applications of PCA could include
identification of novel DNA or RNA binding proteins. In its simplest
conception one uses a known DNA or RNA binding motifs, for instance
a retinoic acid receptor zinc finger, or a simple RNA binding protein such
as IF-1, respectively. One half of the PCA consists of the DNA or RNA
protein binding domain fused to one of the PCA fragments (control
fragment). The complementary fragment is fused to a cDNA library. A
third entity, the gene coding for a sequence containing an element known
to bind to the control protein, and then a second putative or known
regulatory element is coded for after this sequence. A test system
consists of tat/tar elements that control elongation in
transcription/translation of HIV genes. An example application would be
identification of tat binding elements that have been proposed to exist in
eukaryotic genomes and may regulate genes in the same or similar way
to that of HIV genes. (SenGupta D. J. et at. (1996) Proc. Natl. Acad. USA
6, 8496-8501).
EXAMPLE 5
Examples of PCA applications to drug screening: Screening
combinatorial libraries of compounds for those that inhibit or induce
protein-protein/protein-ma/protein-DNA complexes
T"
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A) Drug screening
Screening combinatorial libraries of compounds for
those that inhibit or induce protein-protein/protein-ma/protein-DNA
complexes. The PCA strategy can be directly applied to identifying
potentially therapeutic molecules contained in combinatorial libraries of
organic molecules. It is possible to perform high throughput screening of
such libraries to screen for compounds that will inhibit or induce protein-
protein interactions or protein-DNA/RNA interactions (as discussed
above). In addition it is also possible to screen for compounds that inhibit
enzymes whose substrates are other proteins DNA, RNA or
carbohydrates, as discussed below. In this application, proteins that
interact/protein substrate pairs, or control DNA/RNA binding protein-
enzyme pairs are fused to PCA complementary fragments and plasmids
harboring these pairs are transformed/transfected into a cell, along with
any third DNA or RNA element as the case requires.
Transformed/transacted cells are grown liquid culture in multiwell plates
where each well is inoculated with a single compound from an array of
combinatorially synthesized compounds. A readout of a response
depends on the effect of a compound. If the compound inhibits a protein
interaction, there is a negative response (no PCA signal is the positive
response). If the compound induces a protein interaction, the response
is a positive PCA signal. Controls for non-specific effects of compounds
include: 1) demonstration that the compound does not effect the PCA
enzyme itself (test against cells transfected with the wild-type intact
enzyme used as the PCA probe) and in the case of a cell survival assay,
that the compound is not toxic to the cells that have not been
transformed/transfected. As well as providing a high throughput assay for
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biological activity of compounds, PCA also offers the advantage over in
vitro assays that it is a test for cell membrane permeability of active
compounds. Specific demonstrated examples of PCA for drug screening
in our laboratory include the application of DHFR PCA in E. coli to
detecting compounds that inhibit therapeutically relevant targets. These
include Bax/BcI2 fkbp12/tor ras/raf, carboxyl terminal dimerization
domain of HIV-1 capsid protein, IkB kinase IKK-1 and IKK-2 dimerization
domains (leucine zippers and helix-loop-helix domains). In each case,
the two proteins are subcloned 5' upstream of either F[1,2] or F[3] as
described above. Plasmids harboring the complementary fragments are
cotransformed into BL21 cells. Colonies from minimal medium plates
containing IPTG and trimethoprim are picked, and grown in liquid medium
under the same selective conditions and frozen stocks made. For a
single screening cycle, a priming overnight culture is grown from frozen
stocks in LB medium. A selective minimal medium containing
trimethoprim, ampicillin, IPTG is aliquated at 25 ml into each well of a 384
well plate. Each well is then inoculated with 1 ul of an individual sample
from a compound array (ArQule Inc.) to give a final concentration of 10
uM. Each well is then inoculated with 2 ml of overnight culture and
plates are incubated in a specially adapted shaker bath at 37C. At 2 hour
intervals, plates are read on an optical absorption spectroscopic plate
reader coupled to a PC and spreadsheet software at 600 nm (scattering)
for a period of 8 hours. Rates of growth are calculated from individual
time readings for each well and compared to a standard curve. A "hit" is
defined as a case where an individual compound reduces the rate of
growth to less than the 95 % confidence interval based on the standard
deviation for growth rates observed in all of the wells within the test plate.
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"Near hits" are defined as those cases where growth rates are within the
95 % confidence interval. For each of the hits or near hits, the following
controls are then performed: The same experiment is performed with
BL21 cells that are transformed with empty vector (and no trimethoprim),
with vector harboring the full length mDHFR gene, or with cotransfected
cells where protein expression is not induced by IPTG. If in all of these
cases the compound has no effect, it can be concluded that it is
specifically disrupting the protein-protein interaction being tested. Such
validated hits or near hits are then retested to establish a dose-response
curve for the individual compound, with concentrations varying from 1 pM
up to 1 mM by orders of magnitude of 10. The PCA strategy for
compound screening can also be applied in the multiprotein protein-
RNA/DNA cases as described above, and can easily be adapted to the
DHFR or any other PCA in E. coli or in yeast versions of the same PCAs.
Such screening can also be applied to enzymes whose targets are other
proteins or nucleic acids for known enzyme/substrate pairs or to novel
enzyme substrate pairs identified as described below.
Proteins involved in viral integration processes are examples of targets
that could be tested for inhibitors using the PCA strategies. Examples for
the HIV virus include:
i) Inhibition of integrase or the transport of the pre-integration complex:
protein Ma or vpr.
ii) Inhibition of the cell cycle in G2 by vpr (interaction by cyclin B)
causing
induction of apoptosis.
_
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iii) Inhibition of the interaction of gp160 (precursor of the membrane
proteins) with furine.
Accessory proteins of HIV as a therapeutic target:
i) Vpr: nuclear localizing sequence (target): interaction site of vpr with
phosphatasesA.
ii) vif: interaction with vimentin (cytoskeleton associated protein) .
ii) Vpu: Degradation of CD4 in the RE mediated by the cytoplasmic tail of
Vpu.
iii) net Myristoylation signal of Nef.
Other general targets for drug screening could include
proteins linked neurodegenerative diseases, such as to alpha-synuclein.
This protein has been linked to early onset of Parkinson disease and it is
present also implicated in in Alzheimer disease. There is also b-amyloid
proteins, linked to Alzheimers disease.An example of protein-carbohydrate
interactions that
would be a target for drug screening includes the selectins that are
generally implicated in inflammation. These cell surface glycoproteins are
directly involved in diapedesis. A number of tumor supressor genes whos
actions are
mediated by protein-protein interactions could be screened for potential
anti-cancer compounds. These include PTEN, a tumor supressor directly
involved in the formation of harmatomas. It is also involved in inherited
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breast and thyroid cancer. Other interesting tumor supressor genes
include p53, Rb and BARC1.
EXAMPLE 6
Examples of applications the PCA strategy to detect
enzyme/substrate interactions
The examples described above are used for identifying
novel molecular interactions involving molecules that merely bind to each
other. However detecting the substrates of enzymes is also fully
compatible with the PCA strategy as shown below:
i) Enzymes that form tight complexes or with protein substrates or induce
efficient PCA fragment assembly or
ii) Mutant enzymes that bind tightly to substrate but do not undergo
product release because of mutations residues involved in nucleophilic
attack and/or product release (substrate trapping).
Enzymes may form tight complexes with their substrates
(Kd ¨1-10 mM). In these cases PCA may be efficient enough to detect
such interactions. However, even if this is not true, PCA may work to
detect weaker interactions. Generally, if the rate of catalysis and product
release is slower than the rate of folding- reassembly of the PCA
complementary fragments, effectively irreversible folding and
reconstitution of the PCA reporter activity will have occurred. Therefore,
even if the enzyme and substrate are no longer interacting, the PCA
signal is detected. Therefore, the detection of novel enzyme substrates
using PCA may be possible, independent of effective substrate Kd or rate
_ .
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of product release. In cases where product release is much faster than
PCA fragment assembly/folding and alternative approach is provided by
generating "substrate trapping" mutants of the test enzyme. An example
of this approach applied to the protein tyrosine phosphatase PTP1B,
where substrate trapping mutants have been generated by mutating the
nucleophilic aspartate 181 to alanine rendering the enzyme catalyticly
dead, but capable of forming tight complexes with a known substrate, the
EGF receptor and other unknown proteins (Flint, A. J. et al. (1996) Proc.
Natl. Acad. USA 941680-1685). An application of using PCA to screen
for interacting partners of PTP1B is given as follows. We use the
aminoglycoside kinase (AK)-based PCA in transiently transfected COS
or 293 cells. The substrate trap mutant catalytic domain of PTP1B is
fused to N-terminal complementary fragment of AK, while a C-terminal
fusion of the other AK fragment is made to a cDNA library. Cells are co-
transfected with complementary AK pairs and grown in selective
concentrations of G418. After 72 hours, colonies of surviving cells are
picked and in situ PCR is performed using primers designed to anneal to
3' and 5' flanking regions of the cDNA coding region. PCR amplified
products are then 5' sequenced to identify the gene.
Enzyme inhibitors Screening combinatorial libraries of compounds for
those that inhibit enzyme-PROTEIN substrate complexes either with:
i) Enzymes that form tight complexes with protein substrates or
ii) Mutant enzymes that bind tightly to substrate but do not undergo
product release because of the mutation.
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EXAMPLE 7
Applications of the PCA strateay to protein engineering/evolution
The PCA strategy can be used to generate peptides or
proteins with novel binding properties that may have therapeutic value,
as is done today with phage display technology. It is also possible to
develop enzymes with novel substrate or physical properties for industrial
enzyme development. Two detailed examples of the application of the
PCA strategy to these ends are given below, with additional applications
listed below.
1) Selection of high-affinity, heterodimerizing leucine zipper
sequences (J. Pelletier, K. Arndt, A. Plueckthun and S. Michnick,
manuscript in preparation). The mDHFR PCA, described above, was
used in a scheme for the selection of efficiently heterodimerizing,
designed leucine zippers. It has been proposed that the formation of salt
bridges between positively and negatively charged residues at
complementing 6e6 and 6g6 positions is important in stabilizing leucine
zipper formation, though this view has been contested. In order to help
define the importance of salt-bridge formation at the e and g positions,
two leucine zipper libraries were built. Both are based on the GCN4
leucine zipper sequence, but contain sequence information specific to
either Jun or Fos zippers in order to create heterodimerizing pairs. As
well, the e-1 to e-4 and g-1 to g-4 positions in each library were
randomized to code for positively or negatively charged residues, or
neutral polar residues. These libraries were amplified by PCR and
subcloned into the Z-F[1,2] or Z-F[3] constructs (described above) from
which the GCN4 zipper sequences had been removed. The bacterial
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mDHFR PCA selection was performed on selective solid media, as
described earlier. Colonies were picked and sequenced; sequence
analysis reveals that the distribution of charged or neutral residues at e-g
pairs is not random, but is biased toward pairing of opposite charges, or
pairing of a charged with a neutral residue, rather than same-charge
pairing (see figure 7). We reasoned that better zipper pairing should lead
to an increase in efficiency of DHFR-fragment complementation, resulting
in faster bacterial doubling times (see Table 1 in the mDHFR PCA
description), and undertook a selection/enrichment of the novel zippers
relative to GCN4, as follows. The designed zipper libraries, expressed as
N-terminal fusions to the DHFR F[1,2] or F[3:11144 were cotransformed,
clones were picked, propagated and mixed in selective liquid culture, and
the mix was added in a 1:1 000 000 ratio to clone Z-F[1,2] + Z-F[3:1114A]
(original GCN4 leucine zippers). The mixture was propagated in selective
liquid culture over multiple passages. Restriction analysis shows that
within 4 passages, the population of GCN4-expressing bacteria is
diminishing relative to the novel zipper sequences (data not shown),
indicating that some of the designed zipper-containing clones are
propagated at a higher rate than those containing GCN4. Bacteria from
later passages were plated on selective medium, and individual clones
sequenced to reveal the identity of the most successful designed zipper
pairs (data not shown).
2) Application of PCA to enzyme function and design PCA
Development: Adenosine deaminase (ADA) meets all of the criteria for
a PCA listed above. ADA is a small (-40 kD), and easily purified
monomeric zinc metallo-enzyme and the structure of murine ADA has
1
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been resolved. Several in vitro ADA activity assays have been
developed, involving UV spectrophotometry and stopped-flow fluorimetry.
E. coli ADA catalyzes the irreversible conversion of cytotoxic adenine
nucleosides to non-toxic inosines .
Eukaryotic or prokaryotic cells propagated in the
presence of cytotoxic concentrations of adenosine or adenosine analogs
require ADA to detoxify these compounds. This is the basis of a
dominant-selection strategy used to select for cells expressing a specific
gene in mammalian cells. The ADA gene has also been expressed in
SF3834 E. coli cells which lack a gene coding for endogenous ADA.
When the gene coding for ADA is introduced into ADA- bacterial DNA,
those cells that express ADA are able to survive high concentrations of
added adenosine; those that do not, die . This forms the basis of an in
vivo ADA activity assay.
We chose ADA, principally because it can be used as
a dominant selective marker in mammalian and bacterial cells where the
gene has been knocked out. The reason we choose dominant selective
genes is because in screening for novel protein-protein interactions,
particularly testing for interactions of a .known protein against a library of
millions of independent clones, selection serves to filter for cells that may
show a positive response for reasons having nothing to do with a specific
protein-protein interaction. We will use three test systems of interacting
proteins including leucine zipper-forming sequences, the proteins raf and
p21 and the induced oligomerization system, FK506 binding protein
(FKBP) and mTOR that interact through the macrocyclic immuno-
suppressant compound rapamycin. For all of these systems, we will
construct E. coli and mammalian transient transfection plasmids and
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subclone the test proteins as fusions to ADA complementary fragments.
The primary assay will be survival of SF3834 E. coli cells that have been
transformed with the complementary ADA fragments fused to the test
oligomerization proteins in the presense of toxic concentrations of
adensosine. We will then purify fusion proteins from colonies of and
perform in vitro assays of ADA activity as described below. The utility of
the ADA PCA as a method to identify novel proteins that interact with a
test bait will be performed in mammalian COS-7 and HEK-293T cells
transiently transfected with FKBP fused to one of the ADA fragments and
the other fragment fused to a cDNA library from normal human spleen
containing 106 independent clones. As with the E. coli assay, cells that
survive in a medium containing toxic concentrations of ADA is collected
and isolated plasmids will be testd to identify the gene for the interacting
protein by PCR amplification and chain propagation-termination
techniques.
Structural motifs required for protein function: Determination of the
structural elements required for the enzymatic function of ADA are
investigated through alteration of the structures of the enzyme fragments.
At first, ADA is cut into two separate domains - one responsible for
substrate binding (residues 1-210) and one responsible for catalysis
(residues 211-352). These separate pieces will be attached to known
assembly domains, such as leucine zippers (see example 1 above).
Reassembly will restore activity which will be assessed through detailed
in vitro kinetic analysis of the binding and catalytic properties of the re-
assembled enzyme, using UV spectrophotometry and stopped-flow
fiuorimetry to observe the enzymatic reactions. This system will provide
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another handle on the manipulation of enzyme activity that will afford a
powerful tool for enzymatic mechanism study. For example, the
difference in the kinetic behaviour of the reassembled enzyme on mixing
with the substrate, compared to enzyme reassembled in presence of
substrate (where substrate may already be bound by binding domain) will
allow sophisticated level of study of importance of binding energy to
catalysis. Subsequent point mutations to the functional or assembly
domains of the proteins will then allow a very subtle perturbation and
detailed quantification of the relationship of binding energy to catalysis.
This precise control over the structure and assembly of separate
functional domains of the enzyme will permit very sophisticated enzymatic
structure function studies, the definition of structural motifs and an
understanding of their role in catalysis.
Novel protein catalyst design: The detailed knowledge of the enzyme
mechanism gained through determination of the structural requirements
for catalysis will then be exploited through the combination of these
functional ()building blocks() with the functional motifs responsible for
substrate binding and catalysis in other enzymes, allowing the generation
of novel protein catalysts. For example, the catalytic motif from ADA is
modified to a cytidine-binding motif, creating a novel enzyme with
potentially useful catalytic properties. The activity of these novel
enzymes can easily be assessed through in vivo assays similar to that
of the PCA system, or through in vitro activity assays. Furthermore, the
detailed mechanistic investigation of the resulting enzymes possible with
this system will permit the rational design of each subsequent generation
of catalysts.
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EXAMPLE 8
Examples of applications the PCA strateay to detect molecular
interactions in whole organisms
It is a logical extension of the descriptions of PCA
applications above to the utility of these techniques in whole model
organisms such as drosophila, nematodes, zebra fish and puffer fish, as
examples. The sole differences with other listed examples is that vectors
used would need to be different (for example retroviral vectors) and that
any substrates needed by the PCA would need to be bioavailable, or
detection would need to be performed in situ.
EXAMPLE 9
Examples of applications of the PCA strateay to Gene Therapy
Another important embodiment of the invention is to
provide a means and method for gene therapy of mammalian disease. Of
particular interest is the use of PCA therapeutic for treatment of cancer.
In one embodiment of said PCA gene therapy, a PCA is developed
employing fragments (modular protein units) derived from a protein toxin
for example: Pseudomonas exotoxin, .Diptheria toxin and the plant toxin
gelonin, or other like molecules. For therapy of breast cancer for example,
first a mammalian, retroviral, adenoviral, or eukaryotic artificial
chromosomal (EAC's) genetic construct is prepared that introduces one
fragment of the selected toxin under the control of the promoter for
expression of the erbB2 oncogene. Its is well known that the erbB2
oncogene is overexpressed in breast cancer and adenocarcinoma cells
( D. J. Slamon et. al., Science, 1989, 244, 707). The HER2/neu (c-erbB-
2) proto-oncogene encodes a sub-class 1 185-kDa transmembrane
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protein tyrosine kinase growth factor receptor, p1852. Also, the human
erbB2 oncogene is located on chromosome 17, region q21 and
comprises 4,480 base pairs and p185"' serves as a receptor fora 30-
kDa glycoprotein growth factor secreted by human breast cancer cell lines
( R. Lupu et. Al., Science, 1990, 249, 1152).
The transgene is introduced 'in vivo' or 'ex-vivo' into
target cells employing methods known by those skilled in the art e.g.
homologous recombination to insert transgene into locus of interest via
retroviral, adenoviral or EAC's. A second genetic construct comprising a
fusion gene containing a target DNA that encodes an interacting protein
that interacts with erbB2 oncogene discovered by the PCA process
described in this invention and the "second" fragment of the toxin
molecule. This construct is delivered to the patient by methods known in
the art for example as shown in U.S. Patent Nos. 5,399,346 and
5,585,237.
Transgene expression of the erbB2 oncogene-toxin fragment described
will now be under the control of the constitutive oncogene promoter.
Proliferating tumor cells will thus produce one piece of the toxin attached
as a fusion to the erbB2 oncogene.. In the presence of the second
genetic construct expressing the PCA discovered interacting erbB2
oncogene "interacting protein - toxin fragment" construct then: erbB2
oncogene-toxin fragmentA: interacting protein-toxin fragment B will be
created and induce death of target tumor cells through creation of an
active toxin through Protein Fragment Complementation and thus provide
an efficacious and efficient therapy of said disease.
This can be extended to other diseases and other toxins
employing techniques described and embodied in this invention.
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EXAMPLE 10
Examples of applications the PCA strategy to detect molecular
Interactions in vitro
Any of the PCA strategies described above could be
addapted to in vitro detection. Unlike the in vivo PCAs however,
detection would be performed with purified PCA fragment-fusion proteins.
Such uses of PCA have the potential for use in diagnostic kits. For
example the test DHFR assay described above where the interactiing
domains are FKBP12 and TOR could be used as a diagnostic test for
rapamycin concentrations for use in monitoring dossage in patients
treated with this drug.
As shown above, the instant invention provides:
1) Allow for the detection of protein-protein interactions in vivo or in
vitro.
2) Allow for the detection of protein-protein interactions in appropriate
contexts, such as within a specific organism, cell type, cellular
compartment, or organelle.
3) Allow for the detection of induced versus constitutive protein-protein
interactions (such as by a cell growth or inhibitory factor).
4) To be able to distinguish specific-versus non-specific protein-protein
interactions by controlling the sensitivity of the assay.
5) Allow for the detection of the kinetics of protein assembly in cells.
6) Allow for screening of cDNA libraries for protein-protein interactions.
Further aspects of the invention can be demonstrated
by identifying novel interactions with the enzyme p70S6k, to determine its'
regulation and how separate signaling cascades converge on this
enzyme.
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The PCA method is particularly useful for detection of
the kinetics of protein assembly in cells. The kinetics of protein assembly
can be determined using fluorescent protein systems.
= In a further embodiment of the invention, PCA can be
used for drug screening. The techniques of PCA are used to screen for
drugs that block specific biochemical pathways in cells allowing for a
carefully targeted and controlled method for identifying products that have
useful pharmacological properties.
Although the present invention has been
described herein above by way of preferred embodiments thereof, it can
be modified, without departing from the spirit and nature of the subject
invention as defined in the appended claims.
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Abbreviations: PCA, Protein-fragment Complementation Assay; mDHFR,
murine dihydrofolate reductase; hDHFR, human dihydrofolate reductase;
Z-F[1,2], GCN4 leucine zipper-mDHFR fragment[1,2]; USPS, ubiquitiN-
based split-protein sensor; IPTG, isopropyl-b-D-thiogalactopyranoside;
PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, SDS polyacrylamide
gel electrophoresis.
i I
