U.S. patent application number 10/027770 was filed with the patent office on 2002-10-17 for fusion protein and uses thereof.
Invention is credited to Kirchausen, Tomas, Mayer, Bruce J., Saksela, Kalle.
Application Number | 20020151684 10/027770 |
Document ID | / |
Family ID | 22497709 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020151684 |
Kind Code |
A1 |
Mayer, Bruce J. ; et
al. |
October 17, 2002 |
Fusion protein and uses thereof
Abstract
Two fusion proteins are described. One fusion protein comprises
a protein having a modular protein domain, wherein the domain is
replaced by a single chain antibody. The other fusion protein
contains a binding site that binds to a modular protein binding
domain, wherein a linear epitope in the binding site that binds to
the domain is replaced by at least one copy of an epitope that
binds to the single chain antibody. These fusion proteins, and
nucleic acid encoding them can be used to screen for specific
interaction between two proteins.
Inventors: |
Mayer, Bruce J.; (Tolland,
CT) ; Saksela, Kalle; (Helsinki, FI) ;
Kirchausen, Tomas; (Brighton, MA) |
Correspondence
Address: |
David S. Resnick
NIXON PEABODY LLP
101 Federal Street
Boston
MA
02110
US
|
Family ID: |
22497709 |
Appl. No.: |
10/027770 |
Filed: |
December 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10027770 |
Dec 20, 2001 |
|
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PCT/US00/17929 |
Jun 29, 2000 |
|
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60141896 |
Jun 30, 1999 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
C07K 2319/23 20130101;
C12N 9/1205 20130101; C07K 2319/72 20130101; C07K 2319/42 20130101;
G01N 33/531 20130101; C12N 15/62 20130101; C07K 2319/30 20130101;
C07K 2319/00 20130101; C40B 40/00 20130101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 001/00; C07K
014/00; C07K 017/00 |
Claims
What is claimed:
1. A fusion protein comprising a protein containing a modular
protein binding domain (MPBD), wherein the MPBD is substituted by a
single chain antibody.
2. The fusion protein of claim 1, wherein the MPBD is selected from
the group of domains consisting of src homology 2 (SH2), src
homology 3 (SH3) phosphotyrosine binding (PTB) WW, PDZ, 14.3.3,
WD40, EH and Lim.
3. The fusion protein of claim 1, wherein the protein containing a
MPBD is a tyrosine kinase.
4. The fusion protein of claim 4, wherein the MPBD is src homology
3.
5. A gene encoding the fusion protein of claims 1, 2, 3 or 4.
6. A vector containing the gene of claim 5 operably linked to a
promoter.
7. A fusion protein comprising a protein containing a binding site
that binds to a modular protein binding domain (MPBD), wherein a
linear epitope that binds to the MPBD within the binding site is
substituted by at least one antigenic epitope of 6-20 amino acids
that binds to an antibody.
8. The fusion protein of claim 7, wherein the antigenic epitope
binds to a single chain antibody, wherein said single chain
antibody has been substituted for an MPBD of a protein containing
said MPBD.
9. The fusion protein of claim 8, wherein there are multiple copies
of the antigenic epitope present.
10. The fusion protein of claim 8, wherein there are 3-10 copies of
the antigenic epitope present.
11. A gene encoding the fusion protein of claims 7, 8, 9 or 10.
12. A vector containing the gene of claim 11, operably linked to a
promoter.
13. A cell transformed by the vector of claim 6.
14. The transformed cell of claim 13, further transformed by the
vector of claim 12.
15. A cell co-transfected by the vectors of claims 6 and 12.
16. A library of proteins wherein said proteins contain modular
protein binding domain, and each protein has been fused to by a
single chain antibody.
17. A library of proteins, wherein said proteins each contain a
binding site that binds to a modular protein binding domain (MPBD)
and wherein said proteins have been fused to at least one copy of
an antigenic epitope of 6-20 amino acids that binds to an
antibody.
18. A library of nucleic acid sequences encoding the library of
claim 16.
19. A library of nucleic acid sequences encoding the library of
claim 17.
20. An assay for determining the activity of a protein-protein
interaction, comprising: (a) (1) transforming a cell by the vector
of claim 6, and a second vector, wherein said second vector
contains a gene encoding a fusion protein that has a binding site
that binds to a MPBD, wherein a linear epitope that binds to the
MPBD, wherein said binding site is substituted by at least one copy
of an epitope of 6-20 amino acids that binds to the single chain
antibody that has been substituted by the MPBD of the fusion
protein encoded by the first vector or (2) transforming said cell
by the vector of claim 12, and a second vector containing a gene
encoding a fusion protein containing a MPBD, wherein the MPBD is
substituted by a single claim antibody that binds to the antigenic
epitope expressed by said first vector; (b) culturing the
transformed cell; (c) and comparing the activity to a base line
control; and (d) looking at any change in biological activity to
determine the activity of the protein-protein interaction.
21. The assay of claim 20, wherein the cell used does not express
the protein containing the MPBD of the fusion protein encoded by
the gene in the vector of claim 1.
22. The assay of claim 20, wherein the control constitutes at least
two cells wherein each of said cells is transformed by one of the
two vectors but not the other.
23. An assay for determining the activity of a protein-protein
interaction, comprising: (a) (1) transforming a cell by a vector,
wherein said vector contains a gene encoding a fusion protein that
has a binding site that binds to a modular protein binding domain
(MPBD), wherein a linear epitope that binds to the MPBD in said
binding site is replaced by at least one copy of an antigenic
epitope of 6-20 amino acids that binds to a single chain antibody,
and a vector containing a nucleic acid sequence selected from the
library of claim 18, operably linked to a promoter wherein said
single chain antibody binds to said antigenic epitope, or (2)
transforming said cell by a vector containing a gene encoding a
fusion protein containing a MPBD, wherein the MPBD is substituted
by a single claim antibody that binds to the antigenic epitope, and
a vector containing a nucleic acid sequence selected from the
library of claim 17, operably linked to a promoter, wherein said
antigenic epitope binds to said single chain antibody vector; (b)
culturing the transformed cell; (c) and comparing the activity to a
base line control; and (d) looking at any change in biological
activity to determine the activity of the protein-protein
interaction.
24. An assay for determining molecules that interact with a
protein-protein complex comprising; (a) extracellularly combining
the fusion protein of claim 9, with a fusion protein having a MPBD,
wherein the MPBD has been substituted by a single chain antibody
that specifically binds to at least one copy of the epitope of the
protein of claim 9; (b) adding a test molecule; and (c) comparing
the effect on the binding of the complex with a baseline control of
the complex of the two fusion proteins.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to fusion proteins
containing a single chain antibody (sFv) in place of a modular
protein binding domain, or an antibody epitope in place of the
linear binding epitope for a modular protein binding domain and the
use in an assay system for (1) validating that a protein-protein
interaction causes a specific biological activity, (2) identifying
target molecules capable of affecting those interactions and (3)
identifying the biological activities involved in such
interactions.
BACKGROUND OF THE INVENTION
[0002] A wide range of cellular processes involve protein-protein
interactions. For example, the interaction between a receptor and
its target ligand (such as VEGF.sub.165 Receptor--Neuropilin-1
complexes) as well as intracellular interactions (such as
adaptor-kinase complexes). These interactions can cause signal
transduction or processes governing whether a cell will
proliferate, differentiate, die, adhere, migrate or otherwise
respond to its environment.
[0003] These interactions typically involve the modular protein
binding domains (MPBD) of one protein, which are regions of about
60 to 200 amino acids, and the corresponding binding site of the
second protein. Examples of such domains are SH2 (src homology 2),
SH3 (src Homology 3) and PTB. These domains typically bind to
linear peptide epitopes of about 4-10 amino acid residues on their
binding partners.
[0004] These MPBDs are present in a wide variety of functionally
distinct proteins. The SH2 domain, which binds to phosphorylated
tyrosine residues, appears to be associated with a wide range of
activated growth factors, e.g. epidermal growth factor,
platelet-derived growth factor, fibroblast growth factor, etc.
[0005] Despite the fact that numerous MPBDs and their corresponding
binding sites (BSs) have been identified, however, this typically
does not lead to identification of whether a pair of proteins
actually interact in vivo and whether that interaction is important
for biological activity. This is because the binding interaction
between MPBDs and their corresponding binding sites is not highly
specific, but is only partially specific. Consequently, one protein
can bind with several to hundreds of other proteins with virtually
the same affinity. This lack of specificity has created a
fundamental stumbling block. For example, if a single SH3 domain
can bind with virtually identical affinity to tens or even hundreds
of different proteins in the cell, and a single SH3 binding site
can combine with tens or hundreds of different SH3 domains, which
of these hundreds of potential protein complexes actually results
in a specific in vivo function? For example, if protein A binds
with about equal affinity to proteins 1-200, it would be
exceedingly difficult to determine whether the complex of protein
A-protein 5 and not the complex of protein A-protein55 causes a
specific behavior. Eliminating all 200 proteins from one's test
system, will likely introduce numerous undesired cellular
functional artifacts.
[0006] Thus, trying to determine the specific pair of
protein-protein interactions that results in a biological activity
is typically done indirectly. For instance, one can try to
accomplish this identification negatively, i.e. by preventing the
specific interaction between the proteins to determine if
preventing binding of a set of proteins eliminates a function. This
would be done by altering the MPBD and/or BS to prevent binding.
However, such an alteration will also prevent binding with other
proteins. Therefore that method does not definitively confirm that
the specific biomolecular protein complex under investigation
actually interacts in the cell to result in the function
eliminated. For example, protein A may interact with protein 55 at
one point in a pathway, whereas protein 5 interacts with protein D
at a different point in the pathway. Thus, by altering the binding
sites of proteins A and 5, the function could be lost without the
two proteins having to interact directly with each other.
[0007] It would be desirable to have a protein that can still
perform its native functions, but can bind more specifically to
putative partners. It would also be useful to have a method to
replace the relatively nonspecific interactions between two
proteins with a highly specific interaction, thereby allowing the
two proteins of interest to directly interact without concern for
competing interactions with other proteins in the cell.
[0008] Another indirect approach used are "interaction trap"
systems. For example, the yeast two hybrid approach. In this method
a selectable output such as growth on a selected media, or
metabolism of colorimetric substrates is dependent on
reconstituting a protein-protein interaction with a "bait" protein.
Such a system is limited, in that the selectable biological output
is fixed by the experimental system and proteins are screened for
their ability to bind to the target (bait) protein. Further, one is
typically trying to reconstitute function in a foreign system, e.g.
yeast, as opposed to a mammalian cell system. It would be desirable
to have a system that forces interactions between the proteins and
looks at their functional consequences. It would also be desirable
to have a system that more closely resembles the actual cellular
microenvironment where the protein-protein interactions occurs.
SUMMARY OF THE INVENTION
[0009] We have discovered novel fusion proteins that can be used in
assays to identify protein-protein interactions. One fusion protein
comprises a protein containing a modular protein binding domain
(MPBD), wherein the MPBD is substituted by a single chain antibody.
Preferably, the MPBD is selected from the group of domains
consisting of src homology 2 (SH2), src homology 3 (SH3)
phosphotyrosine binding (PTB), WW, PDZ, 14.3.3, WD40, EH, Lim,
etc.
[0010] For example, such a protein is a tyrosine kinase.
[0011] A second fusion protein comprises a protein containing a
binding site that binds to a modular protein binding domain MPBD,
wherein at least one linear epitope that binds to the MPBD within
the binding site is substituted by an antigenic epitope of 6-20
amino acids that binds to the single chain antibody that has been
substituted for the MPBD. Preferably, the second fusion protein
contains multiple copies of the epitope. For example, 2-20, more
preferably 3-15, still more preferably 4-10 copies of the
epitope.
[0012] Nucleic acid sequences encoding these fusion proteins can be
prepared by known techniques. Preferably these sequences (e.g.,
genes) are contained m vectors and are operably linked to a
promoter.
[0013] These vectors can be used to transform a cell.
[0014] These transformed cells can be used to identify the function
of a protein-protein interaction, to identify a particular protein
involved in an interaction and to study the specific effect of
specific functional domains. In one embodiment there is an assay
for determining the activity of a protein-protein interaction,
comprising:
[0015] (a) transforming a cell by a vector containing a gene
encoding the protein wherein the MPBD site has been substituted by
single chain antibody and a second vector, wherein said second
vector contains a gene encoding a fusion protein that has a binding
site for a MPBD, wherein a linear epitope of 6-20 amino acids that
is bound specifically by the single chain antibody is substituted
for the binding site that binds to MPBD;
[0016] (b) culturing the transformed cell;
[0017] (c) and comparing the activity to a base line control;
and
[0018] (d) measuring changes in activity to determine the activity
caused by that protein-protein interaction.
[0019] Preferably, the cell used does not express the protein
containing the MPBD of the fusion protein encoded by the gene
contained in the transforming vector or the effect of the
interaction is dominant or assayed in a way that does not depend on
the lack of the wild-type counterpart of the engineered gene. The
control can be an untransformed cell, or more preferably cells
transformed with each of the modified fusion proteins, alone, but
not together. By this latter way, one can determine the effect of
expression of each of the proteins on the cell and determine what
effects are dependent on the interaction of the two proteins.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 shows the nucleotide sequence (SEQ ID NO: 1) and the
amino acid sequence (SEQ ID NO:2) of 3DX, a second-generation ScFv
derived from monoclonal antibody 9E10.
[0021] FIGS. 2A and 2B show the antibody binding of antibody 3DX to
a Myc-tagged protein (Myc6 NWASP) for proteins bound to beads
(GSH.sub.pulldown) (FIG. 2A) or from whole cell lysates (FIG.
2B).
DETAILED DESCRIPTION OF THE INVENTION
[0022] We have now discovered that by creating a first fusion
protein wherein a single-chain antibody is substituted for a
modular protein binding domain (MPBD) of sixty to two hundred amino
acids and a second fusion protein wherein the linear epitope of the
corresponding binding site for that modular protein binding domain
has been substituted with at least one copy of an epitope to the
single-chain antibody, one can obtain high affinity protein-protein
binding interactions, to the exclusion of competing reactions.
Moreover, these changes can be tailored so that one does not change
or minimally changes the conformation of the fusion protein in
comparison to the wild type conformation.
[0023] Consequently, one does not have to be concerned about
competition resulting in different interactions between one of the
proteins and an unknown protein or that the change results in a
functionally inactive protein.
[0024] Accordingly, one can now assay for the specific affect that
occurs when protein A binds with protein B. This contrasts with
previous methodologies where in trying to determine the effect of
such an interaction, one was looking at negative effects, i.e.
deleting the MPBD of protein A and the binding site protein of B.
Such an assay could not clearly establish that protein A actually
interacts with protein B. For example, assume protein A interacts
with protein D and protein B interacts with protein F and that the
interaction is in the same biological pathway. Deletions in the
MPBD in protein A would prevent binding with protein E, which could
result in a loss of activity. Similarly a deletion in the binding
site of protein B would prevent its ability to bind with protein E,
thereby also resulting in a loss of activity. However, by looking
at the change in function occurring from the above-mentioned
deletions, one would not know that protein A and protein B did not
actually interact with each other.
[0025] By contrast, with the fusion proteins created by inserting a
single chain antibody in place of the MPBD of the protein A, the
ability of protein A to interact with protein D would be
eliminated. And by inserting at least one antigen epitope in place
of the linear binding site epitope of protein B, the ability of
protein B to interact with protein E is eliminated. Instead only
protein A and protein B can interact with each other. Moreover,
since they act positively, namely by actually interacting one can
look for generation of a function instead of loss of function for
the complex. And by retaining the overall conformation of fusion
protein A and wild type protein A and fusion protein B and wild
type protein B, their activity remains the same as the wild type
proteins. Thus, one can determine what activity actually occurs in
vivo when protein A and protein B interact. We sometimes refer to
this screening methodology as the "Functional Interaction Trap"
(FIT).
[0026] One can transform any cell with vectors containing genes
encoding the fusion proteins. One can then compare these fusion
protein transformed cells with a base line of a control of the same
cell to see the differences that occur m these cells. Preferably,
the genes would be under the control of a promoter that results in
high levels of expression of the fusion protein. Other instances
and sequences that result in enhancing expression can be used. For
example, a HIV TAR element upstream and operably linked to the gene
and a sequence encoding the HIV tat will result in increasing
expression by factors of 103. Other such sequences are known.
[0027] Preferably, one can use a cell line where at least one of
the native proteins being studied is not expressed or minimally
expressed. Such cells may occur naturally or may be engineered.
These cells are referred to herein as "knockout" cells.
[0028] Knockout cells are well known in the art. Many knock out
cells are commercially available from a variety of different
manufacturers. If one desires, one can reduce the level of
endogenous gene expression of a particular protein in a cell i.e.
knock out the gene by using anti-sense, or ribozyme approaches to
inhibit or prevent translation of the protein's mRNA transcripts,
preferably targeting the endogenous promoter; intracellular
targeting of antibodies to the protein to prevent its expression,
triple helix approaches to inhibit transcription of the gene,
targeted homologous recombination to inactivate or knock out the
gene or the endogenous promoter. Preferably one would knock out the
endogenous promoter. See Wagner, R. Nature, 3782:333-335 (1994),
Saver et al., Science, 247:1222-1225 (1990), Helene, C.,
Anti-Cancer Drug Des., 6:569-84 (1991); Helene, C. et al., Ann. NY
Acad. Sci., 660:27-36 (1992); Marasco, W. (U.S. Pat. No.
5,965,374). Alternatively, as mentioned above, there are many
instances where the protein of interest is not being expressed at a
particular time in a cell's cycle or in a particular type of cell.
Such cells can readily be screened for, for example, by looking for
presence of the transcript of the protein using standard detection
means including differential display techniques.
[0029] Additionally, in many cases the phenotype will be dominant
(such as seen with oncogenes, viral proteins, etc), and the
activity caused by the protein-protein interaction will be seen in
cells which still express the two proteins.
[0030] In creating one of the fusion proteins, the MPBD of the
protein containing it is replaced with a single chain antibody
(sFv). sFv's are typically about 250 amino acids in length. Thus,
they can readily be substituted for the MPBD without significantly
affecting the conformation of the protein. One can readily tailor
the specific sFv used based on the general conformation of the
protein, and the specific conformation of the MPBD. Single chain
antibodies are well known in the art (see U.S. Pat. No. 4,946,778)
and also commercially available. Preferred single chain antibodies
that can be used in the present invention include those based on
12CA5, which recognizes the influenza virus hemagglutinin (HA)
epitope, and 9E10, which recognizes an epitope from the Myc
protein. Care should be taken that the epitope that the antibody
binds to does not result in a reaction in the cell that can affect
the behaviors being monitored. This can readily be determined by
known techniques. For example, some epitopes are known to be far
more antigenic than others. Alternatively, one can test a cell by
transforming it with a vector containing a gene encoding the fusion
protein expressing the epitope of interest and determining if this
transformation adversely affects the cell as compared to a control
baseline. By using standard techniques, one can select antibodies
that have the desired characteristics. For example, using phage
display methodologies [Burton, D. R., et al., Proc Natl Acad Sci
USA 88:10134-10137 (1991); Hoogenboom H. R., et al., Immunol Rev
130:41-68 (1992); Winter G., et al., Annu Rev Immunol 12:433-455
(1992); Marks, J. D., et al., J Biol Chem 267:16007-16010 (1992);
Nissim, A., et al., EMBO J 13:692-698 (1994); Vaughan T. J., et
al., Nature Bio 14:304-314 (1996); Marks C., et al., New Eng J Med
335:730-733 (1996)]. For example, very large naive human sFv
libraries have been and can be created to offer a large source or
rearranged antibody genes against a plethora of target molecules.
Smaller libraries can be constructed from individuals with a
particular characteristic such as autoimmune [Portolano S., et al.,
J Immunol 151:2839-2851 (1993); Barbas S. M., et al., Proc Natl
Acad Sci USA 92:2529-2533 (1995)] or infectious diseases [Barbas C.
F., et al., Proc Natl Acad Sci USA 89:9339-9343 (1992); Zebedee S.
L., et al., Proc Natl Acad Sci USA 89:3175-3179 (1992)] in order to
isolate disease specific antibodies. Another such construction
includes the use of human monoclonal antibody producing cell lines.
[Marasco, W. A., et al., Pro Natl Acad Sci USA, 90:7889-7893
(1993); Chen, S. Y., et al., Proc Natl Acad Sci USA 91:5932-5936
(1994). In vitro procedures to manipulate the affinity and fine
specificity of the antigen binding site have been reported
including repertoire cloning [Clackson, T., et al., Nature
352:624-628 (1991); Marks, J. D., et al., J Mol Biol 222:581-597
(1991); Griffiths, A. D., et al., EMBO J 12:725-734 (1993)], in
vitro affinity maturation [Marks, J. D., et al., Biotech 10:779-783
(1992); Gram H., et al., Proc Natl Acad Sci USA 89:3576-3580
(1992)], semi-synthetic libraries [Hoogenboom, H. R., supra;
Barbas, C. F., supra; Akamatsu, Y., et al., J Immunol 151:4631-4659
(1993)] and guided selection [Jespers, L. S., et al., Bio Tech
12:899-903 (1994)]. One can subject an sFv to in vitro mutagenesis
and select for variants having the desired characteristics such as
improved binding.
[0031] For example, one can take the 9E10 and 12CA5 antibody,
subject it to in vitro mutagenesis, screen a library such as a
phage display library. One can test for binding by using an assay
such as an ELISA-based assay system. One can create fusions of the
mutant sFvs to, for example, glutathione S-transferase (GST) and
expressing the GST fusions in a transiently transfected cell line
such as 293T cells along with various proteins tagged with the
desired epitope, e.g., Myc or HA epitopes. The antibodies are
compared against the parent antibody. One antibody, derived from
9E10, displayed dramatically improved binding, termed 3DX (see SEQ
ID NOS 1 and 2 and FIG. 1).
[0032] The novel fusion proteins of the present invention can be
used in a number of ways. For example, in an assay to determine the
biological activity of a specific protein-protein pair.
[0033] The following example is illustrative of the ways one can
look at a protein-protein interaction. The role of adaptor proteins
is to modulate the localization, local concentration and binding
partners of the proteins with which they interact. The fundamental
problem with fully understanding their activity is that they
contain MPBD,--namely, SH2 and SH3 domains--that are promiscuous in
their binding activities (i.e. are not specific). Abl is a kinase
that has a corresponding binding site that is believed to form a
complex with an adaptor protein. Yet, because of the mutual lack of
specificity for specific adaptor-kinase complexes it is difficult
to assess which of the many adaptors that Abl can bind to in vivo
is responsible for the activity of interest. This is because Abl
can bind to the SH3 domains of a range of adaptors. Similarly, the
multiple adaptors that contain the SH3 domains that can bind to the
Abl adaptor-binding site may also bind to numerous proteins other
than Abl.
[0034] By the present invention, the interaction interface between
the SH3 domain of the adaptor and their binding sites on Abl (the
kinase) can be replaced by a synthetic high affinity, specific
interaction i.e. sFv and its .about.10 amino acid epitope.
Preferably the main SH3 domain in an adaptor is replaced with sFv
and at least one of the corresponding sFv epitope is inserted into
an Abl molecule in which the authentic adaptor binding sites have
been ablated. This results in only one interaction: sFv in place of
the SH3 domain of adaptors (e.g. Crk, Nck, Grb2)+sFv epitope-Abl.
Preferably one inserts multiple copies of the epitope to increase
the avidity of interaction between the sFv and the tagged partner.
For example, 2-20 copies. Preferably, 3-15 copies of the epitope.
Still more preferably 4-10 copies of the epitope. Even more
preferably, 4-6 copies.
[0035] The addition of spacers and/or linkers between the epitope
or the sFv and their fusion partner can help maintain a wild-type
conformation. Preferred linkers include neutral amino acid residues
such as Gly. The biological activity of the synthetic/kinase
adaptor pairs are assayed in standard tissue culture models.
Accordingly, the application of the invention allows a recombinant
adaptor and kinase to specifically bind to each other in the
absence of competing interactions.
[0036] In another embodiment, the fusion protein, e.g. synthetic
adaptor proteins, can be based on a sFv that is used to bring
specified functional groups into close proximity to the Abl
catalytic domain. Various functional domains for a wide range of
proteins are well known in the art. Other functional domains can
readily be determined by known means such as the deletion mutant
technique. Virtually any arrangement of functional domains desired
can be prepared. These proteins can be used for a range of
functions, for example to:
[0037] 1. test significance of interaction between a specific pair
of proteins (one with MPBD, one with BS) in vitro, in cultured
cells, or in genetically modified animals.
[0038] 2. test which, of a series of potential protein interaction
partners for a protein of interest identified by a prior screening
assay, such as the yeast two-hybrid assay, is responsible for
biological activity of interest in vitro, in cultured cells, or in
genetically modified animals;
[0039] 3. to identify binding partners for protein of interest
containing MPBD or BS whose binding, directed by sFv/epitope
interactions results in biological activity of interest in cells
(e.g., screen libraries containing sFv or epitope in cells
expressing the protein of interest modified with epitope or
sFv).
[0040] 4. to identify proteins that, when associated with another
protein-based functional group by sFv/epitope-mediated
interactions, leads to biological activity of interest (Functional
groups include subcellular targeting signals, oligomerization
domains, engineered constructs permitting induced aggregation, etc.
These can include functional groups fused to sFv or epitope,
screened with libraries of proteins containing epitope or sFv);
and
[0041] 5. to append functional groups (such as chemical groups,
fluorescent dyes, etc.) to protein or proteins of interest in cells
or in vitro (Such functional groups can be covalently coupled to
synthetic peptide epitope and proteins of interest modified with
sFv by known means).
[0042] More specifically, one can create ectopic SH2 domains. For
example one method of bringing novel functional domains into
proximity with a target protein is by using the N-terminal SH2 of
GAP, which is known to have a high affinity for phosphorylated
p62dok. Thus, one can determine if Abl facilitates phosphorylation
(measurable biological output) of p62dok (second known protein).
Adaptors bind Abl via their SH3 domains, and provide an SH2 domain
to the kinase, which may be useful for phosphorylation of some
proteins by Abl. Using this system novel SH2 domains such as that
of GAP can be brought into close proximity to Abl. In this way, one
can, for example, look at the effect of the specific
phosphorylation of a single protein by a kinase. While kinases can
have profound biological effects, it is not clear what the
consequences of phosphorylation of any single substrate might be.
For example, Abl can transform cells, but the specific substrates
essential for this product are not known. However, with Abl
oncogenic transformation requires a functional SH2 domain [Mayer,
B. J., et al., Mol. Cell. Biol. 12:609-610 (1992). Accordingly, if
the SH2 domain of Abl is replaced with a sFv, the mutant will not
transform cells or phosphorylate substrates efficiently. Then,
using at least one copy of an epitope to the sFv in a putative
protein or a library of proteins, one can identify the protein that
Abl specifically phosphorylated. In this manner, one can evaluate
the biological consequences of phosphorylation of the specific
protein, or to isolate and identify from a library, those
substrates (proteins) displaying a desired property (e.g.,
transformation). Other proteins involved in phosphorylation in
addition to Abl can readily be used.
[0043] Similarly, one can create specific targeting domains such as
the focal adhesion target (FAT) region of FAK. This allows the
testing of effects of relocalization (or targeting) in the absence
of the increased processitivity conferred by an SH2 domain.
[0044] In order to circumvent the lack of specificity of
SH2-phosphopeptide interactions, e.g. an SH2/SH3 adaptor, for
instance the Nck SH3 domains can be directly targeted to
subcellular locations known to directly harbor Nck SH2-binding
sites, allowing for the experimental mimicking of signal-induced
creation of localized binding sites for the Nck SH2 domain without
relocalizing other SH2-containing proteins.
[0045] By appropriate mutagenesis one can alter the local
concentration of Nck SH3 domains on the membrane, mimic clustering
of the tyrosine phosphorylated sites involved in normal stimuli
such as TCR engagement or receptor clustering e.g. Eph
receptor.
[0046] Accordingly one can replace any MPBD, such as the SH3 domain
or domains of for example Nck with the V.sub.L and V.sub.H of a
known antibody. These sFv's can be constructed by standard
techniques for example PCR such as RT-PCR from mRNA isolated from a
monoclonal antibody's hybridoma cells using protocols and reagents
in commercially available kits such as those available from
Pharmacia Biotech supplemented with primers whose design can be
based on published reports. The V.sub.H and V.sub.L, i.e. the sFv
fragments can be designed with 5' and 3' restriction sites allowing
easy fusion of the sFv downstream of a specified site such as
glutathione S-transferase (GST) in a gene, which can be in a
desired expression vector, for example, the mammalian expression
vector pEBG [Mayer, et al., Current Biol., 5:296-305 (1995)]
[0047] Consequently, one can use the fusion sFv/epitope
combinations described here as a screening technology to validate
suspected interactions and to identify functionally important
interations.
[0048] PCR generated clones can be tested for their ability to
co-precipitate the epitope protein, for example, in a transfected
mammalian cell. This permits the identification of the appropriate
single chain antibody clones that have the desirable binding. The
sFv identified can be transferred by PCR based mutagenesis into the
gene encoding the desired clone e.g. Nck, by replacing the MPBD
such as SH3 most important for the protein binding e.g. SH3-2. The
remaining SH3 encoding domains in that gene can be mutated to
prevent interaction with endogenous proteins.
[0049] The corresponding binding motifs in the protein of interest
can also be generated by mutagenesis to have the corresponding
epitope to the single chain antibody in place of the binding
portion of the binding site. For example, Nck binds to Abl. This
binding is mediated by 3 PxxP SH3-binding motifs in Abl located
C-terminal to the catalytic domain. The most important of these
motifs is at position 630 in murine c-Abl. In order to minimally
affect that protein the entire binding region should not be
altered. Instead one mutates the minimal amount necessary.
Preferably, changing the two minor binding sites by site directed
mutagenesis such as substituting Ala residues for Pro. Thereafter,
the antibody's epitope is inserted. For example, the epitope to the
12CA5 antibody is (SEQ ID NO:3) YPYDVPDYA and this can be inserted
within the third PxxP motif to create the desired epitope. The
appropriate changes can readily be made depending on the particular
protein being used. For example Cbl binding to Nck is mediated by
both a large Pro rich region between amino acids 482 and 689 of
human c-Cbl and a short motif PERPPKP (SEQ ID NO:4) between 819 and
827. Thus, one would insert, for example, the Myc or the HA epitope
in place of the second binding site and alter or delete the larger
Pro rich region. These altered proteins can be constructed by a
variety of methods. For example, two step PCR based mutagenesis
using Pfu polymerase to minimize polymerase errors.
[0050] Constructs can be sequenced in the area of primer binding
site to confirm the presence of mutations and to detect any
unforeseen changes. This methodology can be used with virtually any
protein pair.
[0051] Even if one can not readily test protein pairs in a cellular
system to ensure that it is the binding between these two proteins
that is responsible for a particular function, one can look at such
prospective native proteins in any system and tag them and use
deletion mutants to determine which are the most important MPBDs
and binding sites in those two proteins.
[0052] This approach can be adapted for studying a wide range of
protein-protein interactions. For example, the interaction of
cellular proteins with certain viral proteins can have many
undesired affects. For instance it is known that the HIV Nef
protein interacts with cellular protein(s) to reduce immune
function. However the interaction of Nef with particular
protein(s), where binding has functional consequences, are not
currently known because of the lack of specificity in protein
binding. One could take a Nef protein insert a particular epitope
into its binding site and attach a single chain antibody, specific
for the inserted epitopes, to a library of proteins. Thereafter,
one can transform cells with the Nef protein and the library of
different proteins. In this manner one can rapidly identify the
protein that interacts with Nef to result in the function being
sought. Once the particular protein is identified, which can be
done by standard techniques, e.g., by downregulation of immune
function by Nef, one can look for compounds that interact with this
protein. This permits a rational drug design.
[0053] Alternatively, one can take a protein that has a MPBD and
replace that with a specific single chain antibody and have the
unknown library of proteins that preferably contain an epitope that
binds to the MPBD tagged to (fused to) the epitope for the sFv.
Preferably, one would replace the MPBD binding site with the
antibody, but typically the proteins will be tagged randomly. In an
alternative embodiment, the library can be a library of proteins
containing the MPBDs, wherein the proteins are tagged to a single
chain antibody and this library is used to determine the effect of
a protein binding to a protein containing an MPBD binding site that
has been replaced with an antibody epitope. Typically, one will use
a library of nucleic acid sequences encoding the desired proteins.
These nucleic acid libraries will be used to create a vector
library that can be used to transform the desired cells.
[0054] Alternatively, fusing the sFv or the epitopes for the sFv to
various dominant targeting sequences or other functional domains
e.g. membrane localization, nuclear localization, focal adhesions
or cross-linkable membrane domains allows for screening for
libraries of either epitope target fusion proteins or sFv target
antibodies fusion proteins. One can then screen for a specific
biological output in specific cellular compartments. Thereafter,
one can use standard techniques to identify the protein
complex.
[0055] One can also look at cells surface receptors to better
understand cell to cell interaction.
[0056] One can also look at a particular interaction between two
proteins isolated from a cell, for example, to look for molecules
that interact with these proteins.
[0057] As aforesaid, in one embodiment, one can bring various
functional groups in association with a protein of choice. This
requires that one of the two proteins be fused to a larger protein
domain, i.e. the sFv, with the second protein only fused to a small
6-20 amino acid residue epitope, more preferably 6-10 amino acid
residue epitope, and still more preferably 10 amino acid residue
epitope for the single chain antibody. This results in the
association of specific domains without the problem of having to
use large proteins which can place structural and sterical
constraints on the fusion partner.
[0058] Many antibodies and sFv's are known in the art and readily
available. For example, 1287 human V.sub.H sequences and 1041 human
V.sub.L sequences are available in Andrew C. R. Martin's Kabat Man
web page
(http://www.biochem.ucl.ac.uk/.about.martin/abs/simkab.html).
[0059] Alternatively one can readily prepare a desired single chain
antibody. One method is by using hybridoma mRNA or splenic mRNA as
a template for PCR amplification of such genes [Huse, et al.,
Science 246:1276 (1989)]. For example, antibodies can be derived
from murine monoclonal hybridomas [Richardson J. H., et al., Proc
Natl Acad Sci USA 92:3137-3141 (1995); Biocca S., et al., Biochem
and Biophys Res Comm, 197:422-427 (1993) Mhashilkar, A. M., et al.,
EMBO J. 14:1542-1551 (1995)]. These hybridomas provide a reliable
source of well-characterized reagents for the construction of
antibodies and are particularly useful when their epitope
reactivity and affinity has been previously characterized. Another
source for such construction includes the use of human monoclonal
antibody producing cell lines. [Marasco, W. A., et al., Proc Natl
Acad Sci USA, 90:7889-7893 (1993); Chen, S. Y., et al., Proc Natl
Acad Sci USA 91:5932-5936 (1994)]. As discussed above, another
method includes the use of antibody phage display technology to
construct new antibodies against different epitopes on a target
molecule.
[0060] Other sources include transgenic mice that contain a human
immunoglobulin locus instead of the corresponding mouse locus as
well as stable hybridomas that secrete human antigen-specific
antibodies. [Lonberg, N., et al., Nature 368:856-859 (1994); Green,
L. L., et al., Nat Genet 7:13-21 (1994)]. Such transgenic animals
provide another source of human antibody genes through either
conventional hybridoma technology or in combination with phage
display technology. Starting materials for these recombinant DNA
based strategies include RNA from mouse spleens [Clackson, T.,
supra] and human peripheral blood lymphocytes [Portolano, S., et
al., supra; Barbas, C. F. et al., supra; Marks, J. D., et al.,
supra; Barbas, C. F., et al., Proc Natl Acad Sci USA 88: 7978-7982
(1991)] and lymphoid organs and bone marrow from HIV-1-infected
donors [Burton, D. R., et al., supra; Barbas, C. F., et al., Proc
Natl Acad Sci USA 89:9339-9343 (1992)].
[0061] Accordingly, antibody genes can be prepared based upon the
present disclosure by using any known techniques such as those
described.
[0062] Thereafter, using any of these antibodies, one can construct
V.sub.H and V.sub.L genes. For instance, one can create V.sub.H and
V.sub.L libraries from murine spleen cells that have been immunized
either by the above-described in vitro immunization technique or by
conventional in vivo immunization and from hybridoma cell lines
that have already been produced or are commercially available. One
can also use commercially available V.sub.H and V.sub.L libraries.
One method involves using the spleen cells to obtain mRNA which is
used to synthesize cDNA. Double stranded cDNA can be made by using
PCR to amplify the variable region with a degenerative N terminal V
region primer and a J region primer or with V.sub.H family specific
primers, e.g., mouse-12, human-7.
[0063] For example, the genes of the V.sub.H and V.sub.L domains of
the desired antibody such as one to influenza hemaglutin can be
cloned and sequenced. The first strand cDNA can be synthesized
from, for example, total RNA by using oligo dT priming and the
Moloney murine leukemia virus reverse transcriptase according to
known procedures. This first strand cDNA is then used to perform
PCR reactions. One would use typical PCR conditions, for example,
25 to 30 cycles using e.g. Vent polymerase or Pfu polymerase to
amplify the cDNA of the immunoglobulin genes. DNA sequence analysis
is then performed. [Sanger, et al., Proc. Natl. Acad. Sci. USA
79:5463-5467 (1977)].
[0064] Both heavy chain primer pairs and light chain primer pairs
can be produced by this methodology. One preferably inserts
convenient restriction sites into the primers to make cloning
easier. The V.sub.H and V.sub.L chains can be joined together by
convenient linkers, which are known in the art.
[0065] Those of ordinary skill in the art will recognize that a
large variety of possible moieties can be coupled to the resultant
antibodies or to other molecules of the invention for ease in
subsequently identifying a functionally important complex. This is
particularly useful when one is using a library of proteins to
identify the unknown protein that interacts with a known protein.
See, for example, "Conjugate Vaccines", Contributions to
Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds),
Carger Press, New York, (1989), the entire contents of which are
incorporated herein by reference. In some instances, one would use
genetic engineering to couple moieties.
[0066] Coupling may be accomplished by any chemical reaction that
will bind the two molecules so long as the antibody and the other
moiety retain their respective activities. This linkage can include
many chemical mechanisms, for instance covalent binding, affinity
binding, intercalation, coordinate binding and complexation. The
preferred binding is, however, covalent binding. Covalent binding
can be achieved either by direct condensation of existing side
chains or by the incorporation of external bridging molecules. Many
bivalent or polyvalent linking agents are useful in coupling
protein molecules, such as the antibodies of the present invention,
to other molecules. For example, representative coupling agents can
include organic compounds such as thioesters, carbodimides,
succinimide esters, diisocyanates, glutaraldehydes, diazobenzenes
and hexamethylene diamines. This listing is not intended to be
exhaustive of the various classes of coupling agents known in the
art but, rather, is exemplary of the more common coupling agents.
(See Killen and Lindstrom 1984, "Specific killing of lymphocytes
that cause experimental Autoimmune Myasthenia Gravis by
toxin-acetylcholine receptor conjugates." Jour. Immun.
133:1335-2549; Jansen, F. K., H. E. Blythman, D. Carriere, P.
Casella, O. Gros, P. Gros, J. C. Laurent, F. Paolucci, B. Pau, P.
Poncelet, G. Richer, H. Vidal, and G. A. Voisin. 1982.
"Immunotoxins: Hybrid molecules combining high specificity and
potent cytotoxicity". Immunological Reviews 62:185-216; and Vitetta
et al., supra).
[0067] Preferred linkers are described in the literature. See, for
example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984)
describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide
ester). See also, Umemoto et al. U.S. Pat. No. 5,030,719,
describing use of halogenated acetyl hydrazide derivative coupled
to an antibody by way of an oligopeptide linker. Particularly
preferred linkers include: (i) EDC
(1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride;
(ii) SMPT
(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene
(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6
[3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat
#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6
[3-(2-pyridyldithio)pro- pianamide] hexanoate (Pierce Chem. Co.
Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide:
Pierce Chem. Co., Cat. #24510) conjugated to EDC.
[0068] The linkers described above contain components that have
different attributes, thus leading to conjugates with differing
physio-chemical properties. For example, sulfo-NHS esters of alkyl
carboxylates are more stable than sulfo-NHS esters of aromatic
carboxylates. NHS-ester containing linkers are less soluble than
sulfo-NHS esters. Further, the linker SMPT contains a sterically
hindered disulfide bond, and can form conjugates with increased
stability. Disulfide linkages, are in general, less stable than
other linkages because the (disulfide linkage is cleaved in vitro,
resulting in less conjugate available. Sulfo-NHS, in particular,
can enhance the stability of carbodimide couplings. Carbodimide
couplings (such as EDC) when used in conjunction with sulfo-NHS,
forms esters that are more resistant to hydrolysis than the
carbodimide coupling reaction alone.
[0069] Antibodies that are part of the fusion proteins of the
present invention can be detected by appropriate assays, e.g.,
conventional types of immunoassays. For example, a sandwich assay
can be performed in which an antibody to the antibody or a specific
fragment thereof is affixed to a solid phase. Incubation is
maintained for a sufficient period of time to allow the antibody in
the sample to bind to the immobilized antibody on the solid phase.
After this first incubation, the solid phase is separated from the
sample. The solid phase is washed to remove unbound materials and
interfering substances such as non-specific proteins which may also
be present in the sample. The solid phase containing the antibody
of interest bound to the immobilized polypeptide is subsequently
incubated with labeled antibody or antibody bound to a coupling
agent such as biotin or avidin. Labels for antibodies are
well-known in the art and include radionucleotides, enzymes (e.g.
maleate dehydrogenase, horseradish peroxidase, glucose oxidase,
catalase), fluors (fluorescein isothiocyanate, rhodamine,
phycocyanin, fluorescamine), biotin, and the like. The labeled
antibodies are incubated with the solid phase and the label bound
to the solid phase is measured, the amount of the label detected
serving as a measure of the amount of the antibody of interest
present in the sample. These and other immunoassays can be easily
performed by those of ordinary skill in the art.
[0070] The resultant fusion proteins can be expressed by a vector
containing a DNA segment encoding the single chain antibody-MPBD
containing protein described above.
[0071] These can include vectors, liposomes, naked DNA,
adjuvant-assisted DNA, gene gun, catheters, etc. Vectors include
chemical conjugates such as described in WO 93/04701, which has
targeting moiety (e.g. a ligand to a cellular surface receptor),
and a nucleic acid binding moiety (e.g. polylysine), viral vector
(e.g. a DNA or RNA viral vector), fusion proteins such as described
in PCT/US 95/02140 (WO 95/22618) which is a fusion protein
containing a target moiety (e.g. an antibody specific for a target
cell) and a nucleic acid binding moiety (e.g. a protamine),
plasmids, phage, etc. The vectors can be chromosomal,
non-chromosomal or synthetic.
[0072] Preferred vectors include viral vectors, fusion proteins and
chemical conjugates. Retroviral vectors include moloney murine
leukemia viruses. Another vector is a lentiviral vector. Preferably
the lentiviral vector is a pseudotyped lentiviral vector, which
contains heterologous envelope glycoprotein. See, e.g., U.S. Pat.
No. 5,981,276. One can use these fusion proteins to examine the
effect of their interactions in in vivo systems. Thus, vectors can
be selected depending upon the cells where the interaction is being
examined. These vectors include pox vectors such as orthopox or
avipox vectors, herpesvirus vectors such as a herpes simplex I
virus (HSV) vector [Geller. A. I. et al., J. Neurochem, 64: 487
(1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.
Glouer, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A.
I. et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A.
I., et al., Proc Natl. Acad. Sci USA 87:1149 (1990)], Adenovirus
Vectors [LeGal LaSalle et al., Science, 259:988 (1993); Davidson,
et al., Nat. Genet 3:219 (1993); Yang, et al., J Virol. 69:2004
(1995)] and Adeno-associated Virus Vectors [Kaplitt, M. G., et al.,
Nat. Genet. 8:148 (1994)].
[0073] Pox viral vectors introduce the gene into the cells
cytoplasm. Avipox virus vectors result in only a short term
expression of the nucleic acid. Adenovirus vectors,
adeno-associated virus vectors and herpes simplex virus (HSV)
vectors are preferred for introducing the nucleic acid into neural
cells. The adenovirus vector results in a shorter term expression
(about 2 months) than adeno-associated virus (about 4 months),
which in turn is shorter than HSV vectors. The particular vector
chosen will depend upon the target cell that one wants to look at a
specific interaction in. The introduction can be by standard
techniques, e.g. infection, transfection, transduction or
transformation. Examples of modes of gene transfer include e.g.,
naked DNA, CaPo.sub.4 precipitation, DEAE dextran, electroporation,
protoplast fusion, lipofection, cell microinjection, and infection
using viral vectors.
[0074] The vector can be employed to target essentially any desired
target cell, such as a glioma. For example, stereotaxic injection
can be used to direct the vectors (e.g. adenovirus, HSV) to a
desired location. Additionally, the particles can be delivered by
intracerebroventricular (icv) infusion using a minipump infusion
system, such as a SynchroMed Infusion System. A method based on
bulk flow, termed convection, has also proven effective at
delivering large molecules to extended areas of the brain and may
be useful in delivering the vector to the target cell (Bobo et al.,
Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,
Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used
include catheters, intravenous, parenteral, intraperitoneal and
subcutaneous injection, and oral or other known routes of
administration.
[0075] The cell to be tested, e.g. a knock-out cell
(2.times.10.sup.6 cells) can be transfected with DEAE-dextran using
10-14 .mu.g of a vector such as pRc/CMV-sFv containing protein
vector, and incubated at, for example, 37.degree. C. in RPMI media.
48 hours post-transfection, the cells are exposed to selection
media with, for example, 500 .mu.g/ml G418. Six to eight days
later, the bulk stable cells are thoroughly washed with PBS. Cells
are preferably grown at 37.degree. C. in a humidified incubator
with 5% CO.sub.2.
[0076] Preferably one transiently transfects the cells by known
means such as described by Dean, et al., Proc. Natl. Acad. Sci. USA
90:8392-96 (1993).
[0077] Alternatively, the gene encoding the fusion protein
containing the sFv can be cloned into a retroviral vector such as
the LNCX MuLV shuttle vector under the control of the CMVIE
promoter (A. Miller, Central Topics in Microbiology and Immunology,
158 (1991)). The vectors (10 .mu.g) can be transfected by calcium
phosphate into a ecotropic cell line such as PE501 (10.sup.6
cells/100 mm dishes) (A. Miller, Central Topics in Microbiology and
Immunology, 158 (1991)). Twelve hours later, the cells are washed
with PBS and three ml of fresh medium are added to the cells. After
an additional 24 hours, the supernatants from the transfected cells
are collected, cleared by low speed centrifugation (3000.times. g;
1200 rpm), filtered through a 0.45 .mu.m filter and three ml are
used to infect the amphotropic packaging cell line, PG13 (10.sup.6
cells/100 mm dish) in the presence of 8 .mu.g/ml protamine sulfate.
48 hours post-infection, the cells are washed and treated with
selection medium containing 800 .mu.g/ml G418. Once producer cell
lines are established, confluent monolayer cells are split and
fresh medium is added. The cells are then incubated at 32.degree.
C., fusion protein containing supernatants are harvested, filtered
and analyzed. One can transfect the cells with one of the fusion
proteins and then transfect the cell lines with the other fusion
protein or co-transfect the cells with both proteins. With certain
vectors such as herpes virus, HIV, pox virus, the cell can encode
both fusion proteins in the same vector. One can also use transient
transfection by known techniques. [Pear, W., et al., Proc. Nati.
Acad. Sci. USA, 90:8392-8396 (1993). Preferably, one would
transfect the cell in bulk.
[0078] Typically when one is using a library one first transfects
the cells with the gene encoding the known protein. Thereafter, the
bulk cells with the property of interest are selected or wells
containing the transfected cells are transfected by different
proteins from the library. When one is testing a specific protein
pair, one preferably co-transfects the cells or uses a single
vector expressing both proteins.
EXAMPLES
[0079] Human 293% cells were cotransfected with plasmids encoding
GST or GST fused to the parental 9E10 ScFv or the second-generation
derivative, 3DX, with or without plasmids encoding N-WASP tagged
with 6 copies of the Myc tag (Myc.sub.6NWASP) for a single HA tag
(HA-NWASP). FIG. 1 shows the amino acid and nucleotide sequence of
3DX. Amino acid residues that differ from the parental 9E10 sFx are
in bold and underlined, with the corresponding parental 9E10 amino
acids shown below in parenthesis. Two days post-transfection, cells
were lysed in buffer containing 1% Triton X-100 and GST proteins
were recovered by binding to glutathione-agarose beads. Proteins
bound to beads (GSH pulldown) (FIG. 1A) or whole cell lysates (FIG.
2B) were immunoblotted with a polyclonal antibody recognizing
N-WASP. The positions of various N-WASP species are indicated by
arrows.
[0080] All references described herein are incorporated herein by
reference.
* * * * *
References