Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CELL SURFACE DISPLAY OF PROTEINS
BY RECOMBINANT HOST CELLS
TECHNICAL FIELD
l0 The present invention relates generally to methods for expressing
recombinant proteins on the surface of host cells. In particular, the present
invention
relates to strategies for producing a fusion protein comprising a membrane
anchor that
allows extracellular attachment of the fusion protein in a type II
orientation.
BACKGROUND OF THE INVENTION
The expression of foreign proteins on the surface of cells and virus
particles provides a powerful tool for such diverse activities as obtaining
specific
antibodies, determining enzyme specificity, exploring protein-protein
interactions, and
introducing new functions into proteins. Surface display technology is also
used for
expression cloning, in which the biological function of a cloned gene product
is used
for selection.
A number of methods have been devised to display peptides and
proteins on the surfaces of bacteria and bacteriophages. The surface display
of
heterologous protein in bacteria has been implemented for various purposes,
such as
the production of live bacterial vaccine delivery systems (see, for example,
Georgiou
et al., U.S. Patent No. 5,348,867; Huang et al., U.S. Patent No. 5,516,637;
Stahl and
Uhlen, Trends Biotechnol. 15:185 (1995)). Bacterial surface display has been
achieved using chimeric genes derived from bacterial outer membrane proteins,
lipoproteins, fimbria proteins, and flagellar proteins. Bacteriophage display
of foreign
peptides and proteins has become a powerful tool for generating antigens,
identifying
peptide ligands, mapping enzyme substrate sites, isolation of high affinity
antibodies,
and the directed evolution of proteins (see, for example, Phizicky and Fields,
Microbiol. Rev. 59:94 (1995); Kay et al., Phage Display of Peptides and
Proteins
(Academic Press 1996); Lowman, Annu. Rev. Biophys. Biomol. Struct. 26:401 (
1997)).
Either bacterial or bacteriophage surface display systems can be used
for expression screening. Both approaches, however, share certain drawbacks
for
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2
expressing eukaryotic proteins. Prokaryotic cells do not efficiently express
functional
eukaryotic proteins, and these cells lack the ability to introduce post-
translational
modifications, including glycosylation. Moreover, bacterial and bacteriophage
display
systems are limited by the small capacity of the display system, and as such,
are more
suited for the display of small peptides.
There are a limited number of reports on the eukaryotic cell surface
display of heterologous proteins. Boder and Wittrup, Nature Biotechnol. 15:553
( 1997), have described a library screening system using Saccharomyces
cerevisiae as
the displaying particle. This yeast surface display method uses the a-
agglutinin yeast
adhesion receptor, which consists of two subunits, Agal and Aga2. The Agal
subunit
is anchored to the cell wall via a ~3-glucan covalent linkage, and Aga2 is
linked to
Agal by disulfide bonds. In this approach, recombinant yeast are produced that
express Agal and an Aga2 fusion protein comprising a foreign polypeptide at
the C
terminus of Aga2. Agal and the fusion protein associate within the secretory
pathway
~5 of the yeast cell, and are expressed on the cell surface as a display
scaffold.
Various approaches in eukaryotic systems achieve surface display by
producing fusion proteins that contain the polypeptide of interest and a
transmembrane
domain from another protein to anchor the fusion protein to the cell membrane.
In
eukaryotic cells, the majority of secreted proteins and membrane-bound
proteins are
20 translocated across an endoplasmic reticulum membrane concurrently with
translation
(Wicker and Lodish, Science 230:400 (1985); Verner and Schatz, Science
241:1307
( 1988); Hartmann et al., Proc. Nat'l Acad. Sci. USA 86:5786 ( 1989); Matlack
et al.,
Cell 92:381 ( 1998)). In the first step of this co-translocational process, an
N-terminal
hydrophobic segment of the nascent polypeptide, called the "signal sequence,"
is
25 recognized by a signal recognition particle and targeted to the endoplasmic
reticulum
membrane by an interaction between the signal recognition particle and a
membrane
receptor. The signal sequence enters the endoplasmic reticulum membrane and
the
following nascent polypeptide chain begins to pass through the translocation
apparatus
in the endoplasmic reticulum membrane. The signal sequence of a secreted
protein or
30 a type I membrane protein is cleaved by a signal peptidase on the luminal
side of the
endoplasmic reticulum membrane and is excised from the translocating chain.
The rest
of the secreted protein chain is released into the lumen of the endoplasmic
reticulum.
A type I membrane protein is anchored in the membrane by a second hydrophobic
segment, which is usually referred to as a "transmembrane domain." The C-
terminus
35 of a type I membrane protein is located in the cytosol of the cell, while
the N-teminus
is displayed on the cell surface.
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In contrast, certain proteins have a signal sequence that is not cleaved, a
"signal anchor sequence," which serves as a transmembrane segment. A signal
anchor
type I protein has a C-terminus that is located in the cytosol, which is
similar to type I
membrane proteins, whereas a signal anchor type II protein has an N-terminus
that is
located in the cytosol.
Several insect cell systems have been devised to express a fusion
protein comprising a foreign amino acid sequence and a transmembrane domain.
In
one system, an expression vector was designed to allow fusion of a
heterologous
protein to the amino-terminus of the Autographa californica nuclear
polyhedrosis
1o virus major envelop glycoprotein, gp64 (Mottershead et al., Biochem.
Biophys. Res.
Commun. 238:717 ( 1997)). Gp64, a type I integral membrane protein, functions
as an
anchor for the heterologous amino acid sequence, which is displayed on the
surface of
baculovirus particles (Monsma and Blissard, J. Virol. 69:2583 (1995)). More
recently,
Ernst et al., Nucl. Acids Res. 26:1718 (1998), described a baculovirus surface
display
~ 5 system for the production of an epitope library. In this case, a
nucleotide sequence
encoding a particular epitope was inserted into an influenza virus
hemagglutinin gene.
Influenza virus hemagglutinin, like gp64, is a type I integral membrane
protein, which
provides a membrane anchor for the foreign amino acid sequence (see, for
example,
Lamb and Krug, "Orthomyxoviridae: The Viruses and Their Replication," in
2o Fundamental Virology, 3~d Edition, pages 606-647 (Lippincott-Raven
Publishers
1996)).
While both yeast and insect systems are useful for expressing
eukaryotic polypeptides, post-translational modification of mammalian proteins
in
these systems does not necessarily produce proteins that are similar to those
produced
25 by mammalian cells. Accordingly, researchers are interested in developing
display
systems that use mammalian cells.
Cell surface display methods have been used to select molecules that
encode proteins having a signal sequence or a transmembrane domain. For
example,
several techniques rely upon selection for nucleic acid fragments encoding a
signal
30 sequence to identify cDNA molecules that encode secreted proteins or type I
membrane proteins (see, for example, Tashiro et al., Science 261:600 ( 1993);
Yokoyama-Kobayashi et al., Gene 163:193 ( 1995)). According to these methods,
a 5'-
terminal fragment of the test cDNA is fused to a reporter gene, and the
construct is
introduced into cultured cells. If the fusion protein has a functional signal
sequence,
35 the product of the reporter gene will be detected in the cell membrane or
in the culture
medium. Similarly, Davis et al., Science 266:816 (1994), described an
expression
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4
cloning method in which cDNA molecules encoding membrane-bound ligands were
transfected into mammalian cells. Cells that expressed a membrane-bound ligand
of
interest were localized using detestably labeled soluble receptors, and cDNA
encoding
the ligand was rescued from the labeled cells.
In a related selection approach, Yokoyama-Kobayashi et al., Gene
228:161 ( 1999), described a method to test whether a hydrophobic sequence
located
near the N-terminus of a protein functions as a type II signal anchor. Here, a
cDNA
fragment containing the putative type II signal anchor of a target gene was
fused to the
5'-end of a reporter gene. Transfected cells expressed the fusion protein on
the cell
t o surface.
Skarnes et al., Proc. Nat'l Acad. Sci. USA 92:6592 (1995), described a
gene trap method that relies upon capturing the N-terminal signal sequence of
an
endogenous gene to generate an active (3-galactosidase fusion protein, which
is active
in the cytosol, but not in the lumen of the endoplasmic reticulum (also see,
Skarnes,
t5 U.S. Patent No. 5,767,336). Briefly, a vector was designed that expressed a
fusion
protein containing a transmembrane domain of a type I membrane protein and ~i-
galactosidase. The vector was introduced into cultured mammalian cells and
allowed
to integrate into the genome. Insertion of the vector into genes that contain
a signal
sequence produced a fusion protein that is inserted into the endoplasmic
reticulum
20 membrane in a type I configuration. The presence of the signal sequence
results in an
active ~3-galactosidase moiety that is located in the cytosol. In contrast,
insertion of
the vector into a gene that lacks a signal sequence results in a fusion
protein that is
inserted into the endoplasmic reticulum membrane in a type II orientation.
Skarnes et
al. suggested that, in the absence of a signal sequence, the transmembrane
domain of
25 the fusion protein acts a signal anchor sequence. Since the ~3-
galactosidase moiety of
the fusion protein is not located in the cytosol, (3-galactosidase activity is
lost. A
modification of this approach requires an expression vector comprising a
chimeric
gene that contains a secretory lumen-sensitive indicator marker and a type II
secretory
protein transmembrane domain that is positioned N-terminally of the marker
(Skarnes,
3o U.S. Patent No. 5,789,653).
Thus, the methods of Skarnes et al. rely upon the presence of a signal
sequence in the target protein to correct a membrane orientation imposed by an
exogenous transmembrane domain. A foreign transmembrane domain can also be
used to force expression of proteins to the surface of mammalian cells. For
example,
35 Yang, U.S. Patent No. 5,665,590, described a method for cloning genes or
gene
fragments that encode cell surface proteins or secreted proteins. In this
approach, a
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cDNA library is cloned into expression vectors that encode an identifiable
marker and
a membrane anchoring segment. If a cloned cDNA molecule encodes a polypeptide
having a signal sequence, then cells producing the encoded polypeptide should
express
the polypeptide and the identifiable marker as a cell surface protein attached
by the
5 membrane anchoring segment. This method requires the insertion of a cDNA
molecule, which includes an intact 5'-end, upstream of nucleotide sequences
encoding
the identifiable marker and the membrane anchoring segment.
pDisplayTM is an example of a commercially available vector that is
used to display a polypeptide on the surface of a mammalian cell (INVITROGEN
Corp.; Carlsbad, CA). In this vector, a multiple cloning site resides between
sequences that encode two identifiable peptides, hemagglutinin A and myc
epitopes.
The vector also includes sequences that encode an N-terminal signal peptide
derived
from a murine immunoglobulin K-chain, and a type I transmembrane domain of
platelet-derived growth factor receptor, located and the C-terminus. In this
way, a
~ 5 protein of interest is expressed by a transfected cell as an extracellular
fusion protein,
anchored to the plasma membrane at the fusion protein C-terminus by the
transmembrane domain.
Methods that rely upon the selection of certain features, such as a signal
sequence or transmembrane domain, cannot be used to isolate genes encoding all
types
20 of proteins. Moreover, these methods require that the cloned gene or gene
fragment
includes an intact 5'-end that encodes the signal sequence. While more
generally
useful for displaying cloned genes, the pDisplayTM vector has a number of
drawbacks.
For example, the cloned gene will be expressed as an internal segment of a
fusion
protein, which means that both ends of the cloned gene must be inserted in-
frame with
25 the expression vector. Consequently, the vector is most suited for the
display of a
protein encoded by a known nucleotide sequence that can be engineered to
produce the
displayed fusion protein. In addition, the pDisplayTM vector is not well
suited for the
display of representative full-length libraries. This is so because the
polypeptide
encoded by the cDNA must be configured as an internal fusion protein, which
means
30 that the cloned cDNA must not contain the endogenous translation
termination codon,
located at the C-terminus of the gene. The pDisplayTM vector system,
therefore, is best
suited for cloning randomly primed cDNA molecules, which are shorter and are
not
representative of full-length cDNA libraries.
Accordingly, a need still exists for a simple method for expressing any
35 polypeptide, and especially a full-length protein, in a cell surface
display system.
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BRIEF SUMMARY OF THE INVENTION
The present invention provides nucleic acid molecules and methods for
expressing a peptide or polypeptide on the surface of a eukaryotic cell. These
methods
include strategies for producing a fusion protein that comprises a membrane
anchor,
which allows extracellular attachment of the fusion protein in a type II
orientation.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a diagram of the basic components of one type of cell
surface display vector, as described herein. A translation termination signal
or
polyadenylation signal sequence ("Poly(A) site") can be provided by the cloned
gene
or gene fragment.
Figure 2 shows a diagram of an illustrative cell surface display
~5 expression vector. Poly(A) site: polyadenylation signal sequence; TMD:
transmembrane domain; Trans term signal: translation termination signal.
Figure 3 shows a diagram of vector pSLBSDF2-l, which was used to
express thrombopoietin and Arabidoposis thaliana peroxidase, as described in
the
examples. BGH: bovine growth hormone; CMV: cytomegalovirus; Poly(A) site:
20 polyadenylation signal sequence; TMD: transmembrane domain; TNF: tumor
necrosis
factor; Trans term signal: translation termination signal.
DETAILED DESCRIPTION OF THE INVENTION
Zs 1. Overview
The methods described herein provide a means to display a full-length
and post-translationally processed protein encoded by an engineered nucleotide
sequence, or to display a multiplicity of proteins encoded by cloned DNA
molecules,
such as an oligo dT-primed cDNA library, or a random-primed cDNA library. In
3o brief, the display system uses the signal anchor domain sequences of type
II cell
surface proteins to anchor recombinant proteins onto the surface of
transfected cells.
As described above, a characteristic feature of type II cell surface proteins
is that they
are held within the cellular membrane by a single hydrophobic transmembrane
domain
and are oriented with their carboxyl terminus outside the cell. This
orientation is
35 opposite to a type I cell surface protein, in which the N-terminus is
displayed outside
the cells.
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7
One advantage of a display system that uses a type II signal anchor
domain for cell surface attachment is that the recombinant protein can be
produced as
fusion protein having only one fusion junction. This means that one in three
cDNA
molecules will produce an in-frame fusion gene when oligo-dT primed cDNA
molecules are cloned directionally into an expression vector of the present
invention.
In contrast, only one in nine randomly-primed cDNA molecules would produce an
in-
frame fusion protein when the cDNA sequence must be inserted between
nucleotide
sequences that encode a signal sequence and a type I transmembrane domain. In
addition, certain embodiments of the present invention allow the expression of
polypeptides from a gene library regardless of whether or not the genes
include in-
frame endogenous translation termination codons. This feature allows the
display of
full-length proteins encoded by oligo dT-primed cDNA molecules.
Although it is possible to take advantage of histological examination of
fixed transfected cells that express a fusion protein, the presently described
methods
~5 provide the option of examining cloned functional proteins on the surface
of living
cells. The use of live cells not only avoids the risk of protein denaturation
associated
with fixation techniques, but also enables the identification of cells
expressing desired
proteins by cell sorting and similar methods.
As described herein, the present invention provides isolated nucleic
2o acid molecules, comprising, or consisting of, (a) a eukaryotic promoter,
(b) a
nucleotide sequence encoding a type II signal anchor domain segment, and (c) a
cloning site, wherein the isolated nucleic acid molecule comprises elements
(a) to (c)
in a 5' to 3' order. lllustrative promoters include cytomegalovirus promoter,
rous
sarcoma virus promoter, human immunodeficiency virus long terminal repeat
25 promoter, simian virus 40 promoter, and herpes simplex virus thymidine
kinase
promoter. The cloning site of the nucleic acid molecule can be a multiple
cloning site.
In addition, isolated nucleic acid molecules can further comprise a
nucleotide sequence that encodes a spacer peptide, wherein the spacer peptide
encoding nucleotide sequence resides between the type II signal anchor domain
30 encoding nucleotide sequence and the cloning site, and wherein the spacer
peptide
comprises at least ten amino acids. Alternatively, isolated nucleic acid
molecules can
comprise a nucleotide sequence that encodes an affinity tag, wherein the
affinity tag-
encoding nucleotide sequence resides between the type II signal anchor domain-
encoding nucleotide sequence and the cloning site. Moreover, nucleic acid
molecules
35 can comprise both a spacer peptide-encoding nucleotide sequence and an
affinity tag-
encoding nucleotide sequence.
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The present invention also contemplates nucleic acid molecules
comprising at least one of a splice junction and an intron, wherein the intron-
encoding
nucleotide sequence resides between the promoter and the type II signal anchor
domain-encoding nucleotide sequence.
Nucleic acid molecules can further comprise at least one sequence, two,
or three sequences selected from the group consisting of (a) a translation
termination
sequence, (b) a polyadenylation signal sequence, and (c) a transcription
termination
sequence. A nucleic acid molecule that comprises at least two of sequences (a)
- (c)
includes the sequences in the following 5' to 3' order: translation
termination sequence,
polyadenylation signal sequence, and transcription termination sequence.
The present invention further provides isolated nucleic acid molecules,
wherein at least one nucleotide is added or subtracted to the cloning site to
facilitate
the expression of gene fragments in multiple reading frames.
The present invention also contemplates isolated nucleic acid
~ 5 molecules, comprising (a) a eukaryotic promoter, (b) a nucleotide sequence
encoding a
type II signal anchor domain, and (c) a gene or gene fragment, wherein the
isolated
nucleic acid molecule comprises elements (a) to (c) in a 5' to 3' order, and
wherein the
gene or gene fragment resides in-frame with the nucleotide sequence that
encodes the
type II signal anchor domain.
20 Such nucleic acid molecules can further comprise at least one of a
translation termination sequence, which resides in a 3' position relative to
the gene or
gene fragment, a polyadenylation signal sequence, wherein the polyadenylation
signal
sequence is located 3' to the translation termination sequence, and a
transcription
termination sequence, wherein the transcription termination sequence resides
in a 3'
25 position relative to the polyadenylation signal sequence. These translation
termination
sequences, polyadenylation signal sequences, and transcription termination
sequences
can reside within the gene or gene fragment. Isolated nucleic acid molecules
of the
present invention can comprise a type II signal anchor domain-encoding
nucleotide
sequence, which is heterologous with respect to the gene or gene fragment.
30 The present invention also contemplates vectors and expression vectors
comprising such nucleic acid molecules. These vectors can further comprise at
least
one selectable marker gene, and can further comprise at least two origins of
replication, wherein one origin of replication facilitates replication in an
expression
cell type, and wherein a second origin of replication facilitates replication
in an
35 amplification cell type, and wherein the expression cell type is eukaryotic
and the
amplification cell type is prokaryotic.
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The present invention includes recombinant host cells comprising such
vectors and expression vectors. Illustrative host cells include prokaryotic
host cells,
and eukaryotic host cells. Exemplary eukaryotic host cells include mammalian,
avian,
fungal, and insect cells.
The present invention also contemplates methods for selecting nucleic
acid molecules encoding polypeptides, comprising: (a) transfecting an
expression
vector of the present invention into a eukaryotic host cell to produce a
recombinant
host cell, (b) incubating the recombinant host cell under conditions and a
time
sufficient for expression of the gene or gene fragment, and (c) selecting
recombinant
to host cells that comprise the polypeptide product of the gene or gene
fragment on the
cell surface.
The present invention also provides methods for selecting nucleic acid
molecules encoding polypeptides, comprising: (a) incubating recombinant host
cells,
which comprise an expression vector of the present invention, under conditions
and a
t 5 time sufficient for expression of the gene or gene fragment, and (b)
selecting
recombinant host cells that comprise the polypeptide product of the gene or
gene
fragment on the cell surface.
The present invention also contemplates methods for selecting nucleic
acid molecules encoding polypeptides, comprising: (a) obtaining a collection
of genes
20 or gene fragments, (b) cloning the gene or gene fragments into the cloning
site of a
vector or expression vector of the present invention, (c) transfecting the
product of
step (b) into a eukaryotic cell, (d) incubating the transfected cells under
conditions and
a time sufficient for expression of the gene or gene fragment, and (e)
selecting
transfected cells that that comprise the polypeptide product of the gene or
gene
25 fragment on the cell surface.
Other methods for selecting nucleic acid molecules encoding
polypeptides, comprise: (a) cloning a collection of genes or gene fragments
into the
cloning site of a vector or expression vector of the present invention, (b)
incubating
recombinant eukaryotic cells that comprise the product of step (a) under
conditions
3o and a time sufficient for expression of the gene or gene fragment, and (c)
selecting
recombinant cells that that comprise the polypeptide product of the gene or
gene
fragment on the cell surface.
In addition, the present invention provides methods for selecting a
member of a complementary/anti-complementary binding pair, comprising: (a)
cloning
35 a plurality of genes or gene fragments into the cloning site of a vector or
expression
vector of the present invention, wherein the plurality of genes or gene
fragments
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includes a gene or gene fragment that encodes the first member of a
complementary/anti-complementary binding pair (b) transfecting the product of
step
(a) into eukaryotic cells, (c) incubating the transfected cells under
conditions and a
time sufficient for expression of the gene or gene fragment, and (d) selecting
5 transfected cells that that comprise the polypeptide product of the gene or
gene
fragment on the cell surface by exposing the transfected cells to the second
member of
the complementary/anti-complementary binding pair.
In a variation of this approach, a method for isolating a member of a
complementary/anti-complementary binding pair, comprises: (a) incubating
recombinant eukaryotic cells that comprise an expression vector of the present
invention, under conditions and a time sufficient for expression of a gene or
gene
fragment, wherein the gene or gene fragment encodes the first member of a
complementarylanti-complementary binding pair and (b) selecting recombinant
cells
that that comprise the polypeptide product of the gene or gene fragment on the
cell
~ 5 surface by exposing the recombinant cells to the second member of the
complementary/anti-complementary binding pair.
Examples of complementarylanti-complementary binding pairs include
a receptor/ligand pair or an antibody/epitope pair. In certain variations of
such
methods, the second member of the complementary/anti-complementary binding
pair
can mobilized on a solid support. Moreover, the second member of the
complementary/anti-complementary binding pair can be detectably labeled.
Particular methods of the present invention utilize vectors comprising a
gene or gene fragment that comprises genomic DNA or cDNA. Such cDNA can be
synthesized from a primer comprising a poly(dT) sequence or synthesized from
at least
one primer comprising a sequence of random nucleotides.
The present invention also provides kits comprising a nucleic acid
molecule, vector, expression vector, or recombinant host cell, as described
herein.
These and other aspects of the invention will become evident upon
reference to the detailed description and attached drawings. In addition,
various
references are identified below.
2. Definitions
In the description that follows, a number of terms are used extensively.
The following definitions are provided to facilitate understanding of the
invention.
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As used herein, "nucleic acid" or "nucleic acid molecule" refers to
polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid
(RNA),
oligonucleotides, fragments generated by the polymerase chain reaction (PCR),
and
fragments generated by any of ligation, scission, endonuclease action, and
exonuclease
action. Nucleic acid molecules can be composed of monomers that are naturally-
occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring
nucleotides (e.g., a-enantiomeric forms of naturally-occurring nucleotides),
or a
combination of both. Modified nucleotides can have alterations in sugar
moieties
and/or in pyrimidine or purine base moieties. Sugar modifications include, for
t o example, replacement of one or more hydroxyl groups with halogens, alkyl
groups,
amines, and azido groups, or sugars can be functionalized as ethers or esters.
Moreover, the entire sugar moiety can be replaced with sterically and
electronically
similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples
of
modifications in a base moiety include alkylated purines and pyrimidines,
acylated
purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic
acid
monomers can be linked by phosphodiester bonds or analogs of such linkages.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate,
phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,
phosphoranilidate,
phosphoramidate, and the like. The term "nucleic acid molecule" also includes
so-
called "peptide nucleic acids," which comprise naturally-occurring or modified
nucleic
acid bases attached to a polyamide backbone. Nucleic acids can be either
single
stranded or double stranded.
The term "complement of a nucleic acid molecule" refers to a nucleic
acid molecule having a complementary nucleotide sequence and reverse
orientation as
compared to a reference nucleotide sequence. For example, the sequence 5'
ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
The term "contig" denotes a nucleic acid molecule that has a
contiguous stretch of identical or complementary sequence to another nucleic
acid
molecule. Contiguous sequences are said to "overlap" a given stretch of a
nucleic acid
3o molecule either in their entirety or along a partial stretch of the nucleic
acid molecule.
For example, representative contigs to the polynucleotide sequence 5'
ATGGAGCTT
3' are 5' AGCTTgagt 3' and 3' tcgacTACC 5'.
The term "structural gene" refers to a nucleic acid molecule that is
transcribed into messenger RNA (mRNA), which is then translated into a
sequence of
amino acids characteristic of a specific polypeptide. A "gene of interest" can
be a
structural gene.
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"Complementary DNA (cDNA)" is a single-stranded DNA molecule that
is formed from an mRNA template by the enzyme reverse transcriptase.
Typically, a
primer complementary to portions of mRNA is employed for the initiation of
reverse
transcription. Those skilled in the art also use the term "cDNA" to refer to a
double-
s stranded DNA molecule consisting of such a single-stranded DNA molecule and
its
complementary DNA strand. The term "cDNA" also refers to a clone of a cDNA
molecule synthesized from an RNA template.
An "isolated nucleic acid molecule" is a nucleic acid molecule that is not
integrated in the genomic DNA of an organism. For example, a DNA molecule that
encodes a growth factor that has been separated from the genomic DNA of a cell
is an
isolated DNA molecule. Another example of an isolated nucleic acid molecule is
a
chemically-synthesized nucleic acid molecule that is not integrated in the
genome of an
organism. A nucleic acid molecule that has been isolated from a particular
species is
smaller than the complete DNA molecule of a chromosome from that species.
15 A "nucleic acid molecule construct" is a nucleic acid molecule, either
single- or double-stranded, that has been modified through human intervention
to
contain segments of nucleic acid combined and juxtaposed in an arrangement not
existing in nature.
"Linear DNA" denotes non-circular DNA molecules having free 5' and
20 3' ends. Linear DNA can be prepared from closed circular DNA molecules,
such as
plasmids, by enzymatic digestion or physical disruption.
A "promoter" is a nucleotide sequence that directs the transcription of a
structural gene. Typically, a promoter is located in the 5' non-coding region
of a gene,
proximal to the transcriptional start site of a structural gene. Sequence
elements within
25 promoters that function in the initiation of transcription are often
characterized by
consensus nucleotide sequences. These promoter elements include RNA polymerase
binding sites, TATA sequences, CAAT sequences, differentiation-specific
elements
(McGehee et al., Mol. Endocrinol. 7:551 ( 1993)), cyclic AMP response
elements,
serum response elements (Treisman, Seminars in Cancer Biol. 1:47 ( 1990)),
30 glucocorticoid response elements, and binding sites for other transcription
factors,
such as CRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye et
al.,
J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response element binding protein
(Loeken, Gene Expr. 3:253 (1993)) and octamer factors (see, in general, Watson
et al.,
eds., Molecular Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing
35 Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303:1 (1994)).
If a
promoter is an inducible promoter, then the rate of transcription increases in
response to
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an inducing agent. In contrast, the rate of transcription is not regulated by
an inducing
agent if the promoter is a constitutive promoter. Repressible promoters are
also known.
A "core promoter" contains essential nucleotide sequences for
promoter function, including the TATA box and start of transcription. By this
definition, a core promoter may or may not have detectable activity in the
absence of
specific sequences that may enhance the activity or confer tissue specific
activity.
A "regulatory element" is a nucleotide sequence that modulates the
activity of a core promoter. For example, a regulatory element may contain a
nucleotide sequence that binds with cellular factors enabling transcription
exclusively
or preferentially in particular cells, tissues, or organelles. These types of
regulatory
elements are normally associated with genes that are expressed in a "cell-
specific,"
"tissue-specific," or "organelle-specific" manner.
An "enhancer" is a type of regulatory element that can increase the
efficiency of transcription, regardless of the distance or orientation of the
enhancer
~ 5 relative to the start site of transcription.
"Heterologous DNA" refers to a DNA molecule, or a population of
DNA molecules, that does not exist naturally within a given host cell. DNA
molecules heterologous to a particular host cell may contain DNA derived from
the
host cell species (i.e., endogenous DNA) so long as that host DNA is combined
with
20 non-host DNA. For example, a DNA molecule containing a non-host DNA segment
that encodes a polypeptide operably linked to a host DNA segment comprising a
transcription promoter is considered to be a heterologous DNA molecule.
Conversely,
a heterologous DNA molecule can comprise an endogenous gene operably linked
with
a promoter derived from a non-host gene. As another illustration, a DNA
molecule
25 comprising a gene derived from a wild-type cell is considered to be
heterologous DNA
if that DNA molecule is introduced into a mutant cell that lacks the wild-type
gene.
A "polypeptide" is a polymer of amino acid residues joined by peptide
bonds, whether produced naturally or synthetically. Polypeptides of less than
about 10
amino acid residues are commonly referred to as "peptides."
3o A "protein" is a macromolecule comprising one or more polypeptide
chains. A protein may also comprise non-peptidic components, such as
carbohydrate
groups. Carbohydrates and other non-peptidic substituents may be added to a
protein
by the cell in which the protein is produced, and will vary with the type of
cell.
Proteins are defined herein in terms of their amino acid backbone structures;
35 substituents such as carbohydrate groups are generally not specified, but
may be
present nonetheless.
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A peptide or polypeptide synthesized within a cell from a heterologous
nucleic acid molecule is a "heterologous" peptide or polypeptide.
An "integrated genetic element" is a segment of DNA that has been
incorporated into a chromosome of a host cell after that element is introduced
into the
cell through human manipulation. Within the present invention, integrated
genetic
elements are most commonly derived from linearized plasmids that are
introduced into
the cells by electroporation or other techniques. Integrated genetic elements
are
passed from the original host cell to its progeny.
A "cloning vector" is a nucleic acid molecule, such as a plasmid, cosmid,
or bacteriophage, that has the capability of replicating autonomously in a
host cell.
Cloning vectors typically contain one or a small number of restriction
endonuclease
recognition sites that allow insertion of a nucleic acid molecule in a
determinable fashion
without loss of an essential biological function of the vector, as well as
nucleotide
sequences encoding a marker gene that is suitable for use in the
identification and
~ 5 selection of cells transformed with the cloning vector. Marker genes
typically include
genes that provide tetracycline resistance or ampicillin resistance.
An "expression vector" is a nucleic acid molecule encoding a gene that is
expressed in a host cell. Typically, an expression vector comprises a
transcription
promoter, a gene, and a transcription terminator. Gene expression is usually
placed
20 under the control of a promoter, and such a gene is said to be "operably
linked to" the
promoter. Similarly, a regulatory element and a core promoter are operably
linked if the
regulatory element modulates the activity of the core promoter.
A "recombinant host" is a cell that contains a heterologous nucleic acid
molecule, such as a cloning vector or expression vector.
25 "Integrative transformants" are recombinant host cells, in which
heterologous DNA has become integrated into the genomic DNA of the cells.
The term "expression" refers to the biosynthesis of a gene product. For
example, in the case of a structural gene, expression involves transcription
of the
structural gene into mRNA and the translation of mRNA into one or more
polypeptides.
3o The term "secretory signal sequence" denotes a DNA sequence that
encodes a peptide (a "secretory peptide") that, as a component of a larger
polypeptide,
directs the larger polypeptide through a secretory pathway of a cell in which
it is
synthesized. The larger polypeptide is commonly cleaved to remove the
secretory
peptide during transit through the secretory pathway.
35 An "isolated polypeptide" is a polypeptide that is essentially free from
contaminating cellular components, such as carbohydrate, lipid, or other
proteinaceous
WO 01/32894 CA 02390017 2002-05-06 pCT/US00/30238
impurities associated with the polypeptide in nature. Typically, a preparation
of
isolated polypeptide contains the polypeptide in a highly purified form, i.e.,
at least
about 80% pure, at least about 90% pure, at least about 95% pure, greater than
95%
pure, or greater than 99% pure. One way to show that a particular protein
preparation
5 contains an isolated polypeptide is by the appearance of a single band
following
sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein
preparation and Coomassie Brilliant Blue staining of the gel. However, the
term
"isolated" does not exclude the presence of the same polypeptide in
alternative
physical forms, such as dimers or alternatively glycosylated or derivatized
forms.
The terms "amino-terminal" and "carboxyl-terminal" are used herein to
denote positions within polypeptides. Where the context allows, these terms
are used
with reference to a particular sequence or portion of a polypeptide to denote
proximity
or relative position. For example, a certain sequence positioned carboxyl-
terminal to a
reference sequence within a polypeptide is located proximal to the carboxyl
terminus
~5 of the reference sequence, but is not necessarily at the carboxyl terminus
of the
complete polypeptide.
As used herein, the term "type II signal anchor domain," or "type II
transmembrane domain," refers to a hydrophobic amino acid sequence found in
eukaryotic type II integral membrane proteins that, during translation,
targets and
2o anchors a polypeptide in the endoplasmic reticulum membrane with a type II
orientation. The phrase "type II orientation," refers to a protein topology in
which the
N-terminus resides in the cytoplasm, while the C-terminus resides within the
lumen of
the endoplasmic reticulum or on an extracellular cell surface.
A "fusion protein" is a hybrid protein expressed by a nucleic acid
molecule comprising nucleotide sequences of at least two genes. In this way, a
fusion
protein comprises as least two amino acid sequences that are not associated
with each
other in nature. As an illustration, Example Two describes a vector that
expressed a
fusion protein comprising a tumor necrosis factor-a transmembrane domain and a
thrombopoietin moiety.
3o When used to describe a component of an expression vector, the
language "gene or gene fragment" refers to a nucleotide sequence that encodes
a
polypeptide or peptide. The gene or gene fragment can be obtained from genomic
DNA, from cDNA, or by an in vitro synthesis technique (e.g., polymerase chain
reaction, chemical synthesis, and the like).
According to the methods described herein, a nucleic acid molecule
may comprise a nucleotide sequence encoding a type II signal anchor domain and
a
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gene (or gene fragment). If the type II signal anchor domain-encoding sequence
and
the gene (or gene fragment) are derived from different genes, then the type II
signal
anchor domain-encoding sequence is considered to be a heterologous type II
signal
anchor domain-encoding sequence, with respect to the gene (or gene fragment).
An
amino acid sequence produced from such a nucleic acid molecule comprises a
type II
signal anchor domain that is heterologous with respect to the polypeptide or
peptide
encoded by the gene or gene fragment.
Conveniently, an expression vector can be constructed that comprises a
nucleotide sequence encoding a type II signal anchor domain. Figures 2 and 3
provide
examples of such vectors. The isolated type II signal anchor domain is
referred to as a
"type II signal anchor domain segment." The amino acid sequence of a type II
signal
anchor domain segment can be derived from a naturally occurnng polypeptide
(e.g.,
tumor necrosis factor, as illustrated in Figures 2 and 3), or the amino acid
sequence
can be devised following the guidelines discussed below.
The term "affinity tag" is used herein to denote a polypeptide segment
that can be attached to a second polypeptide to provide for purification or
detection of
the second polypeptide or provide sites for attachment of the second
polypeptide to a
substrate. In principal, any peptide or protein for which an antibody or other
specific
binding agent is available can be used as an affinity tag. Affinity tags
include a poly-
histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 ( 1985); Nilsson et
al.,
Methods Enzymol. 198:3 ( 1991 )), glutathione S transferase (Smith and
Johnson, Gene
67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad.
Sci. USA
82:7952 ( 1985)), substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204
( 1988)), streptavidin binding peptide, or other antigenic epitope or binding
domain.
See, in general, Ford et al., Protein Expression and Purification 2:95 ( 1991
). DNA
molecules encoding affinity tags are available from commercial suppliers
(e.g.,
Pharmacia Biotech, Piscataway, NJ).
As used herein, the term "immunomodulator" includes cytokines, stem
cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic
factors,
and synthetic analogs of these molecules. Examples of immunomodulators include
tumor necrosis factor, interleukins, colony stimulating factors, interferons,
stem cell
growth factors, erythropoietin, and thrombopoietin.
The phrase "complement/anti-complement pair" denotes non-identical
moieties that form a non-covalently associated, stable pair under appropriate
conditions. For instance, biotin and avidin (or streptavidin) are prototypical
members
of a complementJanti-complement pair. Other exemplary complemendanti-
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complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or
epitope) pairs, sense/antisense polynucleotide pairs, and the like.
An "antibody fragment" is a portion of an antibody such as F(ab')2,
F(ab)2, Fab', Fab, and the like. Regardless of structure, an antibody fragment
binds with
the same antigen that is recognized by the intact antibody.
The term "antibody fragment" also includes a synthetic or a genetically
engineered polypeptide that binds to a specific antigen, such as polypeptides
consisting
of the light chain variable region, "Fv" fragments consisting of the variable
regions of
the heavy and light chains, recombinant single chain polypeptide molecules in
which
light and heavy variable regions are connected by a peptide linker ("scFv
proteins"), and
minimal recognition units consisting of the amino acid residues that mimic the
hypervariable region.
A "detectable label" is a molecule or atom which can be conjugated to
a polypeptide to produce a molecule useful for identifying cells that express
the
binding partner of the polypeptide. Examples of detectable labels include
chelators,
photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or
other
marker moieties.
Due to the imprecision of standard analytical methods, molecular
weights and lengths of polymers are understood to be approximate values. When
such
a value is expressed as "about" X or "approximately" X, the stated value of X
will be
understood to be accurate to ~10%.
3. Design of Expression Vectors
Expression vectors that are suitable for production of a protein in
eukaryotic cells typically contain ( 1 ) prokaryotic DNA elements coding for a
bacterial
replication origin and an antibiotic resistance marker to provide for the
growth and
selection of the expression vector in a bacterial host, (2) eukaryotic DNA
elements that
control initiation of transcription, such as a promoter, and (3) DNA elements
that
control the processing of transcripts, such as a transcription
termination/polyadenylation signal sequence.
An expression vector of the present invention comprises, in a 5' to 3'
direction, a eukaryotic promoter, a signal anchor domain of a type II protein,
and a
nucleotide sequence that is a cloning site, which allows insertion of a gene
or gene
fragment. In addition, the expression vector can also include translation
termination,
polyadenylation signal, and transcription termination sequences, although;
such
elements may be provided by the polypeptide-encoding gene or gene fragment.
The
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18
expression vector can also include a nucleotide sequence that encodes an
affinity tag.
An affinity tag-encoding sequence can be positioned, for example, between the
type II
signal anchor domain-encoding sequence and the cloning site. The expression
vector
can also contain a nucleotide sequence that encodes a spacer peptide, which
can also
be located between the type II signal anchor domain-encoding sequence and the
cloning site. Studies have shown that the presence of an intron can increase
the
efficiency of recombinant protein expression. Accordingly, an expression
vector of
the present invention can include an intron sequence located, for example,
between the
promoter and the type II signal anchor domain-encoding sequence. Expression
vectors
to can also contain additional elements such as a gene that encodes a
selectable marker,
an antibiotic resistance gene for selection in a bacterial host, an SV40 early
promoter
and origin, which drives expression of the selectable marker gene and allows
episomal
replication in cells containing SV40 large T antigen, a ColEl origin, which
provides
replication and growth in E. coli, and the like.
~5 The expression vectors described herein can be used for a variety of
applications. For example, antigen display on the surface of cells can be used
to
modulate immune functions (see, for example, Cho et al., J. Immunol. Meth.
220:179
( 1998)). The display of an otherwise secreted protein or non-secreted protein
on the
cell surface is also useful for studying the interaction between a
complement/anti-
20 complement pair. As an illustration, the examination of the interaction
between a
receptor-ligand pair provides an approach to rational drug design. The
expression
vectors can be used to clone unknown members of a complement/anti-complement
pair. For example, a labeled probe consisting of a known member of a receptor-
ligand
pair can be used to screen cells transfected with a cell surface display cDNA
library.
25 The positive cell can be identified by direct binding of the probe to its
partner
expressed on the cell surface. The cDNA encoding the unknown partner can then
be
recovered from the recombinant host cells. Alternatively, the labeled probe
can be
used as a cell sorting reagent to enrich for a population of library
transfected cells
expressing an interacting partner to the probe.
3o In addition, various bioactive proteins can be displayed on the cell
surface to produce a cell with new useful functions or properties. Bioactive
reactive
molecules include chemo-attractants, adhesion molecules, antigens, antibodies,
enzymes, growth factors, receptors, and the like. The expression of exogenous
proteins on the cell surface can also be used as a live recombinant vaccine.
35 The display of polypeptides on the surface of a recombinant cells can
be used to deliver bioactive molecules to other cells. This mode of delivery
has the
WO 01/32894 CA 02390017 2002-05-06 pCT/US00/30238
19
advantage that the activity is confined to the cell surface, resulting in an
activity that is
exerted locally and specific only to nearby cells. Since the fusion protein
products are
not secreted, the specific activity of the fusion protein is not reduced by
dilution of the
medium.
The cell surface display system can be used to characterize and identify
polypeptides, or peptides, that mediate cell differentiation and growth. For
example,
cDNA molecules encoding test polypeptides can be displayed on the surface of
mammalian cells, which are co-cultured with embryonic stem cells. Under co-
culture
conditions, the recipient cells displaying the test polypeptides are
incorporated into
embryoid bodies formed by the stem cells. Active polypeptides are identified
by the
ability of the recipient cells to induce growth and differentiation of
embryoid body
cells. As another illustration, the display system described herein can
produce a
collection of recipient cells, each of which displays a polypeptide encoded by
a cDNA
from a cDNA library. When co-cultured with stem cells, cDNA molecules encoding
t5 active polypeptides can be identified. cDNA molecules encoding active
polypeptides
that affect growth or differentiation can also be identified by displaying
polypeptides
encoded by complex cDNA libraries directly on the surface of stem cells.
A. Expression Vector Components
20 To express a gene, a nucleic acid molecule encoding the protein must be
operably linked to regulatory sequences that control transcriptional
expression and then,
introduced into a host cell. In addition to transcriptional regulatory
sequences, such as
promoters and enhancers, expression vectors can include transcriptional and
translational
regulatory sequences. As an illustration, the transcriptional and
translational regulatory
25 signals suitable for a mammalian host may be derived from viral sources,
such as
adenovirus, bovine papilloma virus, simian virus, or the like, in which the
regulatory
signals are associated with a particular gene that has a high level of
expression.
Suitable transcriptional and translational regulatory sequences also can be
obtained
from mammalian genes, such as actin, collagen, myosin, and metallothionein
genes.
30 Suitable transcriptional regulatory sequences include a promoter region
sufficient to direct the initiation of RNA synthesis. lllustrative eukaryotic
promoters
include the promoter of the mouse metallothionein I gene (Hamer et al., J.
Molec.
Appl. Genet. 1:273 ( 1982)), the TK promoter of Herpes virus (McKnight, Cell
31:355
( 1982)), the SV40 early promoter (Benoist et al., Nature 290:304 ( 1981 )),
the Rous
35 sarcoma virus promoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777
(1982)),
the cytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and the
mouse
CA 02390017 2002-05-06
WO 01!32894 PCT/US00/30238
mammary tumor virus promoter (see, generally, Etcheverry, "Expression of
Engineered Proteins in Mammalian Cell Culture," in Protein Engineering:
Principles
and Practice, Cleland et al. (eds.), pages 163-181 (John Wiley & Sons, Ine.
1996)).
Alternatively, a prokaryotic promoter, such as the bacteriophage T3
5 RNA polymerase promoter, can be used to control expression of the gene of
interest in
mammalian cells if the prokaryotic promoter is regulated by a eukaryotic
promoter
(Zhou et al., Mol. Cell. Biol. 10:4529 ( 1990), and Kaufman et al., Nucl.
Acids Res.
19:4485 ( 1991 )).
The signal anchor domain component of an expression vector of the
present invention can be any type II signal anchor domain sequence, which is
capable
of providing attachment to the cell surface in a type II orientation. Examples
of type II
cell surface proteins that comprise such signal anchor domains include
influenza
neuraminidase, the small hydrophobic proteins of the paramyxovirus simian
virus, the
paramyxovirus hemagglutinin-neuraminidase, human and rat asialoglycoprotein
~5 receptors, chicken hepatic lectin, human and rabbit neutral endopeptidase,
human
intestinal aminopeptidase, rabbit sucrase-isomaltase receptor, human
transferrin
receptor, hepatic glycoprotein receptor, human IgE receptor, murine 1,4-(3-
galactosyltransferase, human P-glycoprotein receptor, human invariant chains
of class
II histocompatibility antigens, rat sodium channel proteins, rat brain, muscle
and liver
20 glucose transporter proteins, bacterial leader peptidase, and members of
the tumor
necrosis factor/nerve growth factor superfamily (see, for example, Wolfe et
al., J. Biol.
Chem. 258:12073 ( 1983); Chiacchi and Drickamer, J. Biol. Chem. 259:15440 (
1984);
Hiebert et al., J. Virol. 54:1 (1985); Hiebert et al., J. Virol. 55:744
(1985); Schneider
et al., Nature 311:675 ( 1984); Spiess and Lodish, Proc. Nat'! Acad. Sci. USA
82:6465
( 1985); Strubin et al., EMBO J. 3:869 ( 1984); Semenza, Annu. Rev. Cell Biol.
2:255
( 1986); Lipp and Dobberstein, J. Cell Biol. 106:1813 ( 1988); Hartmann et
al., Proc.
Nat'! Acad. Sci. USA 86:5786 ( 1989)). Moreover, Chou and Elrod, Proteins:
Structure, Function, and Genetics 34:137 ( 1999), disclose 152 type II
membrane
proteins, which they used to devise a method for predicting whether an amino
acid
sequence confers the type II membrane protein structure.
The illustrative pSLBSDF2-I vector contains a nucleotide sequence that
encodes the type II signal anchor domain of human tumor necrosis factor-oc
(see Figure
3). Tumor necrosis factor-a (TNF-a) exists as a type II membrane bound
precursor
which is cleaved and released by a converting enzyme, and its signal anchor
domain
sequence is well defined (Utsumi et al., J. Biol. Chem. 268:9511 (1993);
Utsumi et al.,
Molec. Cell. Biol. 15:6398 ( 1995); Tang et al., Biochem. 35:8226, ( 1996);
Moss et al.,
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21
Nature 385:733 ( 1997); Rosendahl et al., J. Biol. Chem. 272:24588 ( 1997)).
The
converting enzyme cleavage site is also well defined (see, for example, Tang
et al.,
Biochem. 35:8226 ( 1996)). The type II signal anchor domain in pSLBSDF2-1
lacks a
cleavage site to prevent the release of displayed protein from the cell
surface. The
illustrative pSLBSDF2-1 vector includes a TNF-a transmembrane domain (signal
anchor domain) with the following amino acid sequence: LFLSL FSFLI VAGAT
TLFCL LHFGV I (SEQ ID N0:2). Preferably, the vector also includes a TNF-a N-
terminus sequence (MSTES MIRDV ELAEE ALPKK TGGPQ GSRRC; SEQ ID
N0:3) positioned at the N-terminal end of the transmembrane domain.
A nucleic acid molecule that encodes a synthetic sequence with
functional properties of a type II signal anchor domain can also be used for
the
expression vectors of the present invention. A synthetic type II signal anchor
domain
sequence can be constructed based on the known functional requirements (see,
for
example, Parks and Lamb, Cell 64:777 (1991)). Studies indicate that the
balance
~5 between the length of the hydrophobic segment and N-terminal charge is
important for
the orientation of cell surface proteins. For example, Sakaguchi et al., Proc.
Nat'l
Acad. Sci. USA 89:16 (1992), found that hydrophobic segments consisting of 7-
10
leucine residues function as type II signal sequences, whereas segments with
12-15
leucine residues showed different topogenic functions, behaving as a signal
sequence
20 or type II signal anchor domain sequence depending on the net charge on the
N-
terminal. In the type II surface proteins, about 90% have a net positive
cytoplasmic
charge in the 15-residue transmembrane-flanking region of the non-translocated
amino
terminus (Hartmann et al., Proc. Nat'l Acad. Sci. USA 86:5786 ( 1989)). Lipp
and
Dobberstein, J. Cell Biol. 106:1813 ( 1988), indicate that a type II signal
anchor
25 domain has three distinct segments: ( 1 ) a net positively-charged N-
terminal region, (2)
a central segment of hydrophobic amino acid residues, containing at least 16
amino
acid residues, and (3) a hydrophilic C-terminal portion.
Alternatively, a signal sequence may be modified to be functionally
equivalent to a type II or a type I signal anchor domain for use in the
expression
30 vectors described herein. Modifications include: (a) an increase in the
length of the
hydrophobic segment to enhance membrane anchorage, (b) increasing or
decreasing
net charge to control orientation within the membrane, and (c) the removal of
cleavage
site for a signal peptidase (see, for example, Chou and Kendall, J. Biol.
Chem.
265:2873 ( 1990); Nilsson et al., J. Cell Biol. 126:1127 ( 1994); Parks, J.
Biol. Chem.
35 271:7187 (1996)).
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22
The inclusion of an affinity tag is useful for the identification or
selection of cells displaying the fusion protein. Examples of affinity tags
include
polyHistidine tags (which have an affinity for nickel-chelating resin), c-myc
tags (e.g.,
EQKLI SEEDL; SEQ ID N0:4) which are detected with anti-myc antibodies,
calmodulin binding protein (isolated with calmodulin affinity chromatography),
substance P, the RYIRS tag (which binds with anti-RYIRS antibodies), a
hemagglutinin A epitope tag (e.g., YPYDV PDYA; SEQ ID N0:5) which is detected
with an antibody, the Glu-Glu tag, and the FLAG tag (which binds with anti-
FLAG
antibodies). See, for example, Luo et al., Arch. Biochem. Biophys. 329:215 (
1996),
1o Morganti et al., Biotechnol. Appl. Biochem. 23:67 (1996), and Zheng et al.,
Gene
186:55 ( 1997). Nucleic acid molecules encoding such peptide tags are
available, for
example, from Sigma-Aldrich Corporation (St. Louis, MO).
The cloning site can be a multicloning site. Any multicloning site can
be used, and many are commercially available. Particularly useful multicloning
sites
allow the cloning of a gene or gene fragment in all three reading frames.
The expression vector can also include a transcription termination
sequence, and optionally, a polyadenylation signal sequence. For example,
pSLBSDF2-1 includes a bovine growth hormone polyadenylation signal sequence
and
transcription termination sequence to enhance mRNA stability. An expression
vector
2o need not contain transcription termination and polyadenylation signal
sequences,
because these elements can be provided by the cloned gene or gene fragment.
As shown in Figure 3, pSLBSDF2-I includes two sets of three-frame
termination codons, one set located 3' to an EcoRI site, and one set located
5' to a XhoI
site. The first set of termination codons can be used for cDNA molecules
cloned into
the EcoRI site. The second set of termination codons can be used for cDNA
molecules that are cloned directionally as EcoRI-XhoI fragments. Such DNA
molecules can be produced, for example, by random priming.
The expression vector can include a nucleotide sequence that encodes a
selectable marker. A wide variety of selectable marker genes are available
(see, for
3o example, Kaufman, Meth. Enzymol. 185:487 ( 1990); Kaufman, Meth. Enzymol.
185:537
(1990)). For example, one suitable selectable marker is a gene that provides
resistance
to the antibiotic neomycin. In this case, selection is carned out in the
presence of a
neomycin-type drug, such as G-418 or the like. Bleomycin-resistance genes,
such as
the Sh ble gene, are also useful selectable marker genes for the presently
described
methods. These genes produce a protein that inhibits the activity of
bleomycin/phleomycin-type drugs, such as ZEOCIN (Gatignol et al., Mol. Gen.
Genet.
CA 02390017 2002-05-06
WO 01/32894 PCT/US00/30238
23
207:342 (1987); Drocourt et al., Nucl. Acids Res. 18:4009 (1990)). ZEOCIN is
toxic
in a broad range of cell types, including bacteria, fungi, plant, avian,
insect, and
mammalian cells. Additional selectable markers include hygromycin B-
phosphotransferase, the AURI gene product, adenosine deaminase, aminoglycoside
phosphotransferase, dihydrofolate reductase, thymidine kinase, and xanthine-
guanine
phosphoribosyltransferase (see, for example, Srivastava and Schlessinger, Gene
103:53 (1991); Romanos et al., "Expression of Cloned Genes in Yeast," in DNA
Cloning 2: Expression Systems, 2"d Edition, pages 123-167 (IRL Press 1995);
Markie,
Methods Mol. Biol. 54:359 ( 1996); Pfeifer et al., Gene 188:183 ( 1997);
Tucker and
Burke, Gene 199:25 ( 1997); Hashida-Okado et al., FEBS Letters 425:117 (
1998)).
Selectable marker genes can be cloned or synthesized using published
nucleotide
sequences, or marker genes can be obtained commercially.
A expression vector can also include an SV40 origin. This element can
be used for episomal replication and rescue in cell lines expressing SV40
large T
IS antigen.
The expression vectors of the present invention can express any nucleic
acid molecule encoding an amino acid sequence of interest as a fusion protein
comprising a type II signal anchor domain. Typically, the type II signal
anchor domain
and the amino acid sequence of interest are not associated with each other in
nature,
20 and therefore, are heterologous with respect to each other. That is, these
two amino
acid sequences typically are encoded by nucleotide sequences of different
naturally-
occurring genes.
Exemplary amino acid sequences of interest include full-length
polypeptides, and fragments of full-length polypeptides. Although the cloned
gene or
25 gene fragment can encode a peptide, the gene or gene fragment preferably
encodes a
polypeptide comprising more than 10 amino acids. For example, such
polypeptides
can consist of about 10 to about 20 amino acids, about 20 to about 40 amino
acids,
about 40 to about 100 amino acids, or greater than 100 amino acids.
A gene or gene fragment suitable for insertion into an expression vector
30 can be obtained from cDNA, which is prepared by any method known in the
art. For
example, cDNA molecules can be synthesized by random priming. Moreover, such
primers can be linked to restriction endonuclease sites found in the vector.
Alternatively, cDNA molecules can be prepared by oligo d(T) priming. A gene or
gene fragment can also be obtained from genomic DNA or by chemical synthesis.
35 Standard methods for preparing suitable genes or gene fragments are known
to those
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24
in the art (see, for example, Ausubel et al. (eds.), Short Protocols in
Molecular Biology,
3'~ Edition (John Wiley & Sons 1995) ["Ausubel 1995"]).
After constructing the expression vector, the vector can be propagated
in a host cell to synthesize nucleic acid molecules for the generation of a
nucleic acid
polymer. Vectors, often referred to as "shuttle vectors," are capable of
replicating m at
least two unrelated expression systems. To facilitate such replication, the
vector
should include at least two origins of replication, one effective in each
replication
system. Typically, shuttle vectors are capable of replicating in a eukaryotic
system
and a prokaryotic system. This enables detection of protein expression in
eukaryotic
l0 hosts, the "expression cell type," and the amplification of the vector in
the prokaryotic
hosts, the "amplification cell type." As an illustration, one origin of
replication can be
derived from SV40, while another origin of replication can be derived from
pBR322.
Those of skill in the art know of numerous suitable origins of replication.
Vector propagation is conveniently carried out in a prokaryotic host
cell, such as E. coli or Bacillus subtilus. Suitable strains of E. coli
include
BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DHI, DH4I, DHS, DHSI, DHSIF',
DHSIMCR, DH l OB, DH l OB/p3, DH 11 S, C600, HB 101, JM 101, JM 105, JM 109,
JM 110, K38, RR 1, Y 1088, Y 1089, CSH 18, ER 1451, and ER 1647 (see, for
example,
Brown (ed.), Molecular Biology Labfax (Academic Press 1991)). Suitable strains
of
Bacillus subtilus include BR151, YB886, MI119, MI120, and B170 (see, for
example,
Hardy, "Bacillus Cloning Methods," in DNA Cloning: A Practical Approach,
Glover
(ed.) (IRL Press 1985)). Standard techniques for propagating vectors in
prokaryotic
hosts are well-known to those of skill in the art (see, for example, Ausubel
1995; Wu
et al., Methods in Gene Biotechnology (CRC Press, Inc. 1997)).
B. Expression Vector Variations
Expression vectors can be designed to comprise two "transcriptional
units," in which a transcriptional unit comprises a transcriptional regulatory
element, a
coding region, and a transcription terminator. One coding region would encode
the
polypeptide of interest, while the second coding region would encode the
selectable
marker. Both transcriptional units may contain the same transcriptional
regulatory
element.
As an illustration, Examples 1 and 2 describe studies with an
expression vector, designated as ' pSLBSDF2-I ," which includes two
transcriptional
units. One transcriptional unit comprises a cytomegalovirus (CMV) promoter and
intron which are operably linked with a nucleotide sequence encoding a tumor
CA 02390017 2002-05-06
WO 01/32894 PCT/US00/30238
necrosis factor-a signal anchor domain, a nucleotide sequence that encodes an
affinity
tag, a nucleotide sequence that encodes a 13 amino acid residue spacer
consisting of
glycine and alanine residues to provide spatial freedom to the displayed
protein, a
cloning site, and termination and polyadenylation signal sequences. In the
illustrative
5 vector, the spacer has the following amino acid sequence: GGGGA AGGGG GAA
(SEQ ID NO:1). A second transcriptional unit comprises an SV40 origin and
promoter
operably linked to a neomycin resistance gene. The pSLBSDF2-I vector also
includes
an ampicillin resistance gene and a ColEl origin for selection and propagation
in E.
coli.
A spacer offers the advantages of providing flexibility, and minimal
steric interference with the folding or function of other portions of the
fusion protein.
Those of skill in the art can devise suitable spacers, which meet the
requirement of an
inert, flexible amino acid sequence. For example, a proline residue can be
added to
the illustrative spacer (SEQ ID NO: l ) at the beginning, at the end, or at
both the
~5 beginning and the end of the spacer. In the latter case, the proline
residue would serve
to isolate the spacer as a separate functional domain from the other parts of
the
protein. Such proline residues need not occur at the precise endpoints of the
spacer.
For example, proline residues can be inserted between one to four amino acid
residues
from the spacer endpoints. Moreover, spacers can be devised that include any
of
2o glycine, serine, and alanine residues, and that include from 10 to 30 or
more amino
acid residues. For example, suitable spacers can consist of 25 amino acid
residues to
provide spatial freedom to the displayed protein.
Alternatively, an expression vector can comprise two coding regions,
which reside between a transcriptional regulatory element and a transcription
25 terminator. In this case, each of the coding regions of the dicistronic
message vector
should have its own ribosome binding site (see, for example, Lee et al., Nucl.
Acids
Res. 12:6797 ( 1984)). For example, the second coding sequence of a
dicistronic
vector can encode a reporter protein used to identify a transfected cell that
expresses
the foreign genes. Illustrative reporter proteins include cell surface
proteins that can
be bound with antibodies to isolate cells with a fluorescent activated cell
sorter, or
other method. Another example of a reporter protein is an enzyme that
catalyzes the
formation of a detectable product from a suitable substrate. Moreover, the
reporter
protein itself may be detectable using its inherent physical properties, such
as
fluorescence or light emission.
Another approach accounts for gene or gene fragments that encode a
polypeptide comprising a signal sequence. Proteins that span the cell membrane
more
CA 02390017 2002-05-06
WO 01/32894 PCT/US00/30238
26
than once, the so-called "multipass transmembrane proteins," comprise
transmembrane segments having orientations determined by the most N-terminal
transmembrane domain (see, for example, Hartmann et al., Proc. Nat'l Acad.
Sci. USA
86:5786 ( 1989); Sato et al., J. Biol. Chem. 273:25203 ( 1998)). Transmembrane
domains that follow this first domain alternate in orientation as the
polypeptide spans
the membrane. Multipass transmembrane proteins are illustrated by the seven
transmembrane domain G-protein coupled receptors. Fusion proteins with a type
II
transmembrane domain at its N-terminus, followed by an even number of
transmembrane domains or a functionally equivalent hydrophobic amino acid
sequence (e.g., a signal sequence) would display the remaining portion of the
protein
outside the cell. A vector, designated as "pSLSD-2," was designed to display
protein
containing an endogenous signal peptide sequence. pSLSD-2 is constructed by
the
insertion of a nucleotide sequence encoding a transmembrane domain upstream
(5'
ward) of the cloning site of pSLBSDF2-1 to orient the protein with an
endogenous
signal sequence outside the cell.
4. Production of Recombinant Protein by Host Cells
The expression vector can be introduced into any eukaryotic cell, such as
a mammalian cell, insect cell, avian cell, fungal cell, and the like. Examples
of
2o suitable mammalian host cells include African green monkey kidney cells
(Vero;
ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573),
baby hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL
10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells
(CHO-K1; ATCC CCL61; CHO DG44 (Chasin et al., Som. Cell. Molec. Genet.
12:555, 1986)), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC
CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548) SV40-transformed monkey
kidney cells (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3;
ATCC CRL 1658).
The baculovirus system provides an efficient means to introduce cloned
genes of interest into insect cells. Suitable expression vectors are based
upon the
Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), and
contain
well-known promoters such as Drosophila heat shock protein (hsp) 70 promoter,
Autographa californica nuclear polyhedrosis virus immediate-early gene
promoter
(ie-1 ) and the delayed early 39K promoter, baculovirus p10 promoter, and the
Drosophila metallothionein promoter. A second method of making recombinant
baculovirus utilizes a transposon-based system described by Luckow (Luckow, et
al.,
CA 02390017 2002-05-06
WO 01/32894 PCT/US00/30238
27
J. Virol. 67:4566 ( 1993)). This system, which utilizes transfer vectors, is
sold in the
BAC-to-BAC kit (Life Technologies, Rockville, MD). This system utilizes a
transfer
vector, PFASTBAC (Life Technologies) containing a Tn7 transposon to move the
gene or gene fragment into a baculovirus genome maintained in E. coli as a
large
plasmid called a "bacmid." See, Hill-Perkins and Possee, J. Gen. Virol. 71:971
( 1990), Bonning, et al., J. Gen. Virol. 75:1551 ( 1994), and Chazenbalk, and
Rapoport,
J. Biol. Chem. 270:1543 ( 1995). These vectors can be modified following the
above
discussion
The recombinant virus or bacmid is used to transfect host cells.
Suitable insect host cells include cell lines derived from IPLB-Sf 21, a
Spodoptera
frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL 1711), Sf2lAE, and
Sf21
(Invitrogen Corporation; San Diego, CA), as well as Drosophila Schneider-2
cells,
and the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni (U.S.
Patent
No. 5,300,435). Commercially available serum-free media can be used to grow
and to
~5 maintain the cells. Suitable media are Sf900 IlT"" (Life Technologies) or
ESF 921T""
(Expression Systems) for the Sf9 cells; and Ex-ce11O405TM (JRH Biosciences,
Lenexa,
KS) or Express FiveOT"" (Life Technologies) for the T. ni cells. When
recombinant
virus is used, the cells are typically grown up from an inoculation density of
approximately 2-5 x 105 cells to a density of 1-2 x 106 cells at which time a
20 recombinant viral stock is added at a multiplicity of infection (MOI) of
0.1 to 10, more
typically near 3.
Established techniques for producing recombinant proteins in
baculovirus systems are provided by Bailey et al., "Manipulation of
Baculovirus
Vectors," in Methods in Molecular Biology, Volume 7: Gene Transfer and
Expression
25 Protocols, Murray (ed.), pages 147-168 (The Humana Press, Inc. 1991), by
Patel et al.,
"The baculovirus expression system," in DNA Cloning 2: Expression Systems, 2nd
Edition, Glover et al. (eds.), pages 205-244 (Oxford University Press 1995),
by
Ausubel (1995) at pages 16-37 to 16-57, by Richardson (ed.), Baculovirus
Expression
Protocols (The Humana Press, Inc. 1995), and by Lucknow, "Insect Cell
Expression
30 Technology," in Protein Engineering: Principles and Practice, Cleland et
al. (eds.),
pages 183-218 (John Wiley & Sons, Inc. 1996).
The expression vectors described herein can also be used to transfect
fungal cells, including yeast cells. Yeast species of particular interest in
this regard
include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
Suitable
35 promoters for expression in yeast include promoters from GALL (galactose),
PGK
(phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOXI (alcohol
oxidase),
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28
HIS4 (histidinol dehydrogenase), and the like. Many yeast cloning vectors
readily
available and can be modified following the above discussion. These vectors
include
YIp-based vectors, such as YIpS, YRp vectors, such as YRp 17, YEp vectors such
as
YEpl3 and YCp vectors, such as YCpl9. Methods for transforming S. cerevisiae
cells with exogenous DNA and producing recombinant polypeptides therefrom are
disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,31 l, Kawasaki et
al., U.S.
Patent No. 4,931,373, Brake, U.S. Patent No. 4,870,008, Welch et al., U.S.
Patent No.
5,037,743, and Murray et al., U.S. Patent No. 4,845,075. Transformed cells are
selected by phenotype determined by the selectable marker, commonly drug
resistance
or the ability to grow in the absence of a particular nutrient (e.g.,
leucine). A preferred
vector system for use in Saccharomyces cerevisiae is the POTI vector system
disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows
transformed
cells to be selected by growth in glucose-containing media. Additional
suitable
promoters and terminators for use in yeast include those from glycolytic
enzyme genes
~5 (see, e.g., Kawasaki, U.S. Patent No. 4,599,311, Kingsman et al., U.S.
Patent No.
4,615,974, and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase
genes.
See also U.S. Patents Nos. 4,990,446, 5,063,154, 5,139,936, and 4,661,454.
Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactic, Kluyveromyces
20 fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia
guillermondii
and Candida maltosa are known in the art. See, for example, Gleeson et al., J.
Gen.
Microbiol. 132:3459 (1986), and Cregg, U.S. Patent No. 4,882,279. Aspergillus
cells
may be utilized according to the methods of McKnight et al., U.S. Patent No.
4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by
25 Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming
Neurospora are
disclosed by Lambowitz, U.S. Patent No. 4,486,533.
For example, the use of Pichia methanolica as host for the production
of recombinant proteins is disclosed by Raymond, U.S. Patent No. 5,716,808,
Raymond, U.S. Patent No. 5,736,383, Raymond et al., Yeast 14:11-23 (1998), and
in
3o international publication Nos. WO 97/17450, WO 97/17451, WO 98/02536, and
WO
98/02565. DNA molecules for use in transforming P. methanolica will commonly
be
prepared as double-stranded, circular plasmids, which are preferably
linearized prior to
transformation. For polypeptide production in P. methanolica, it is preferred
that the
promoter and terminator in the plasmid be that of a P. methanolica gene, such
as a P.
35 methanolica alcohol utilization gene (AUGl or AUG2). Other useful promoters
include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase
CA 02390017 2002-05-06
WO 01/32894 PCT/US00/30238
29
(FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the
host
chromosome, it is preferred to have the entire expression segment of the
plasmid
flanked at both ends by host DNA sequences. For large-scale, industrial
processes
where it is desirable to minimize the use of methanol, it is preferred to use
host cells in
which both methanol utilization genes (AUGI and AUG2) are deleted. For
production
of secreted proteins, host cells deficient in vacuolar protease genes (PEP4
and PRBI )
are preferred. Electroporation is used to facilitate the introduction of a
plasmid
containing DNA encoding a polypeptide of interest into P. methanolica cells.
P.
methanolica cells can be transformed by electroporation using an exponentially
decaying, pulsed electric field having a field strength of from 2.5 to 4.5
kV/cm,
preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40
milliseconds,
most preferably about 20 milliseconds.
An expression vector can be introduced into host cells using a variety of
standard techniques including calcium phosphate transfection, liposome-
mediated
~ 5 transfection, microprojectile-mediated delivery, electroporation, and the
like.
Standard methods for introducing expression vectors into mammalian,
yeast, and insect cells are provided, for example, by Ausubel ( 1995). General
methods
for expressing and recovering foreign protein produced by a mammalian cell
system are
provided by, for example, Etcheverry, "Expression of Engineered Proteins in
20 Mammalian Cell Culture," in Protein Engineering: Principles and Practice,
Cleland et
al. (eds.), page 163 (Wiley-Liss, Inc. 1996). Established methods for
isolating
recombinant proteins from a baculovirus system are described by Richardson
(ed.),
Baculovirus Expression Protocols (The Humana Press, Inc. 1995).
Expression vectors can be isolated from cells that produce a
25 polypeptide of interest. If desired, expression vectors can be subjected to
another
round of selection based on expression of the identifiable polypeptide or,
transfected
into the amplification cell type. The transfected amplification cell type is
then selected
by the selectable marker, the vectors are purified and the nucleotide sequence
of the
gene or gene fragment is sequenced by any method known in the art. If the
nucleotide
30 sequence encodes only a portion of a complete polypeptide, then the
nucleotide
sequence can be used as a probe by methods known in the art to retrieve the
entire
gene.
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5. Identification of Recombinant Host Cells That Express Fusion
Protein and Recovery of Nucleic Acid Molecules Encoding the
Fusion Protein
There are various approaches to identifying recombinant host cells that
5 express a polypeptide of interest on the extracellular surface. For example,
the
recombinant host cells can be cultured for a time sufficient to express the
fusion
protein on the cell surface. These cells are then combined with a reagent that
specifically binds to the fusion protein, and that is labeled with a
detectable tag.
Suitable reagents in this regard include antibodies, ligands, soluble
receptors and the
like. Detectable tags suitable for use include fluorescent, fluorescence
quenching, dye
and magnetic tags and the like. In addition, any tag that modifies the light
scattering
properties of the target to which it is bound is suitable for use herein. The
recombinant host cells are then sorted according to the presence or absence of
detectable tag/reagent bound at the cell surface. Thus, in one step,
recombinant host
~5 cells expressing a fusion protein are readily segregated from those in
which a
functional signal sequence is lacking. In one preferred embodiment, an
automated
machine that permits single cell examination (e.g., a flow cytometer) is used
to detect
and to select recombinant host cells that express a fusion protein at the cell
surface. As
an illustration, a fluorescence-activated flow cytometer is used to segregate
cells that
2o express a fusion protein.
The detectably labeled reagent can be used in either soluble form or
bound to a solid support. The phrase "solid support" refers to any material
capable of
binding a member of a complementary/anti-complementary binding pair. Well-
known
solid supports include glass, polystyrene, polypropylene, polyethylene,
dextran, nylon,
25 amylases, natural and modified celluloses, polyacrylamides, agaroses, and
magnetite.
The solid support can have virtually any possible structural configuration so
long as
the bound reagent molecule is capable of binding with a fusion protein. Thus,
the
support configuration may be spherical, as in a bead (e.g., a magnetic bead),
or
cylindrical, as in the inside surface of a test tube, or the external surface
of a rod.
3o Alternatively, the surface may be flat, such as a sheet, a test strip, and
the like. Those
skilled in the art are aware of many other suitable solid supports.
Following identification or selection, the type II signal anchor domain
nucleotide sequence can be used as a probe or as a PCR primer to recover
sufficient
amounts of the DNA of interest for sequencing. As an alternative, selected
recombinant host cells can be cloned and expanded before DNA recovery with a
probe
or PCR primer. After the mixture of DNA molecules of interest is amplified, in
one
alternative, the recovered DNA can be recloned into the expression vector for
WO 01/32894 CA 02390017 2002-05-06 pCT~S00/30238
31
additional cycles of enrichment. After enrichment, individual DNA clones can
be
isolated for sequencing. In another alternative, the mixture of amplified DNA
molecules can be used as a sense primer to generate full-length DNA molecules
of
interest. This library of full-length DNA molecules can then be subjected to
clonal
isolation to obtain a single DNA molecule. Each cloned DNA molecule can then
be
sequenced, expressed, and characterized.
The present invention also contemplates compositions packaged as kits
for producing recombinant host cells that express a fusion protein on the cell
surface. As
used herein, the term "package" refers to a solid matrix or material
customarily
utilized for a kit and capable of holding one or more of the reagent
components for use
in a method of the present invention. Packages can include containers, such as
glass
and plastic (e.g., polyethylene, polypropylene, polycarbonate, etc.) bottles,
vials,
paper, plastic and plastic-foil laminated envelopes, and the like.
A kit comprises at least one container comprising a nucleic acid
~5 molecule, which is a cell surface display expression cassette. An
illustrative cell
surface display expression cassette is a nucleic acid molecule, which
comprises, in a S'
to 3' order: ( 1 ) a eukaryotic promoter, (2) a nucleotide sequence encoding a
type II
signal anchor domain, and (3) a cloning site. The expression cassette can also
comprise a nucleotide sequence (located, for example, between the type II
signal
2o anchor domain and the cloning site) that encodes an affinity tag. Such
expression
cassettes can be included as a component of an expression vector.
The kit can also comprise a second container comprising one or more
reagents capable of indicating the presence of an expressed fusion protein.
For
example, a container can comprise an antibody, or antibody fragment, which
binds
25 with an affinity tag. The antibody or antibody fragment can be detectably
labeled, or a
detectable label can be provided in another container. Additional containers
can
provide reagents for producing a cDNA library.
The reagents can be provided in solution, as a liquid dispersion or as a
substantially dry powder. For example, nucleic acid molecules, antibodies, or
3o antibody fragments can be provided in lyophilized form. A solid support and
one or
more buffers can also be included as separately packaged elements in this
system.
A kit can also comprise a means for conveying to the user that the
reagents are used to produce recombinant host cells expressing a fusion
protein on the
cell surface. The written material can be applied directly to a container, or
the written
35 material can be provided in the form of a packaging insert.
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32
The present invention, thus generally described, will be understood more
readily by reference to the following examples, which are provided by way of
illustration
and are not intended to be limiting of the present invention.
EXAMPLE 1
Cell Surface Display of Thrombopoietin
Thrombopoietin, a four-alpha-helix cytokine, was used to test the
ability of the expression system to display complex, correctly folded,
assembled
proteins on the cell surface. The sequence encoding mature thrombopoietin was
cloned as an EcoRI and XhoI fragment in surface display vector pSLBSDF2-1 in
the
correct reading frame to yield the plasmid pSLBSDF2-TPO. Following the
transfection of pSLBSDF2-TPO into BHK570 or COS- I cells, functional
thrombopoietin was detected on the cell surface, as shown by specific binding
with a
~ 5 horseradish peroxidase-labeled thrombopoietin receptor. Bound horseradish
peroxidase was detected using the TSA-Direct kit, sold by NEN Life Science
Products
(Boston, MA). Briefly, adherent transfected cells were rinsed with phosphate-
buffered
saline to remove any autofluorescent particles, and the cells were incubated
with
diluted fluorescein tyramide for five minutes. The cells were then rinsed with
2o phosphate-buffered saline to remove excess reagent, and the presence of
activated
fluorophor on cell surfaces was visualized with an inverted fluorescent
microscope at
a wavelength of 494 nm excitation/517 nm emission.
These studies included the use of three protocols prior to fluorescein
tyramide treatment. In one protocol, cells were fixed with formaldehyde and
treated
25 with Triton-X to permeabilize cell membranes. To limit detection to the
cell surface, a
second protocol eliminated Triton-X treatment. In a third protocol, both
fixation and
permeabilization steps were omitted.
In addition to cells transfected with pSLBSDF2-TPO, another set of
cells was transfected with a thrombopoietin expression plasmid, in which the
type II
30 signal anchor domain was replaced with a secretion leader. These cells
exhibited
thrombopoietin activity in the cell-conditioned media, but cell surfaces
lacked any
detectable binding of the thrombopoietin receptor.
The detection of functional thrombopoietin on the cell surface of
transfected cells demonstrated that the display system is capable of producing
35 correctly folded and assembled protein, and that the protein is tethered on
the cell
surface in a manner that can be recognized by a receptor.
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33
EXAMPLE 2
Cell Surface Display of Arabidoposis thaliana Peroxidase
In another set of experiments, a transfection vector, designated as
"pSLBSDF2-AP," was constructed to express Arabidoposis thaliana peroxidase.
This
enzyme is a plant peroxidase which requires a heme prosthetic group for
activity. The
sequence encoding the peroxidase was cloned as an EcoRI and XhoI fragment in
surface display vector pSLBSDF2-1. Transfection of BHK 570 or COS-7 cells with
pSLBSDF2-AP resulted in cells with cell surface peroxidase activity, as
determined
using the TSA-Direct kit, described above.
Cell surface deposition of activated fluorescein tyramide was not
observed in expression vectors that were designed to secrete recombinant
Arabidoposis thaliana peroxidase into the culture media. The detection of
functional
~5 Arabidoposis thaliana peroxidase on the cell surface of pSLBSDF2-AP-
transfected
cells showed that the display system is capable of producing correctly folded
and
assembled protein, and that the protein is tethered on the cell surface in a
manner that
can exhibit enzymatic activity.
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1
SEQUENCE LISTING
<110> Lok. Si
<120> Cell Surface Display of Proteins by
Recombinant Host Cells
<130> 99-34
<160> 5
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Peptide spacer.
<400> 1
Gly Gly Gly Gly Ala Ala Gly Gly Gly Gly Gly Ala Ala
1 5 10
<210> 2
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Transmembrane domain
<400> 2
Leu Phe Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr
1 5 10 15
Leu Phe Cys Leu Leu His Phe Gly Val Ile
20 25
<210> 3
<211> 30
<212> PRT
CA 02390017 2002-05-06
WO 01/32894 PCT/US00/30238
2
<213> Artificial Sequence
<220>
<223> N-terminal sequence
<400> 3
Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala
1 5 10 15
Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys
20 25 30
<210> 4
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> c-Myc tag
<400> 4
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 5
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Hemagglutinin A epitope tag
<400> 5
Tyr Pro Tyr Asp Ual Pro Asp Tyr Ala
1 5
