U.S. patent application number 11/048574 was filed with the patent office on 2005-06-16 for method of identifying a gene product.
This patent application is currently assigned to GenVec, Inc.. Invention is credited to Carrion, Miguel E., Kovesdi, Imre, McVey, Duncan L..
Application Number | 20050130219 11/048574 |
Document ID | / |
Family ID | 26910732 |
Filed Date | 2005-06-16 |
United States Patent
Application |
20050130219 |
Kind Code |
A1 |
Carrion, Miguel E. ; et
al. |
June 16, 2005 |
Method of identifying a gene product
Abstract
The present invention provides a method of identifying a gene
product. The method comprises providing a multiplicity of cells
comprising a first gene product. Preferably, the first gene product
is produced in the multiplicity of cells by expressing a first
exogenous nucleic acid sequence encoding the first gene product. A
library of second nucleic acid sequences encoding second gene
products is then introduced into the multiplicity of cells. The
second nucleic acid sequences are expressed in the multiplicity of
cells to produce the second gene products such that the first gene
product and at least one of the second gene products contact. The
method further comprises causing a complex to form between the
first gene product, an affinity molecule that binds the first gene
product, and at least one of the second gene products, and
subsequently retrieving the complex. At least one second gene
product of the complex then is identified.
Inventors: |
Carrion, Miguel E.; (New
Market, MD) ; Kovesdi, Imre; (Rockville, MD) ;
McVey, Duncan L.; (Derwood, MD) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6780
US
|
Assignee: |
GenVec, Inc.
Gaithersburg
MD
|
Family ID: |
26910732 |
Appl. No.: |
11/048574 |
Filed: |
February 1, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11048574 |
Feb 1, 2005 |
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10336702 |
Jan 3, 2003 |
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6861229 |
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10336702 |
Jan 3, 2003 |
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PCT/US01/21445 |
Jul 6, 2001 |
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60272943 |
Mar 2, 2001 |
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60216174 |
Jul 6, 2000 |
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Current U.S.
Class: |
435/6.11 ;
435/456; 435/5 |
Current CPC
Class: |
C12Q 1/6897 20130101;
C12N 15/1055 20130101; C12Q 2563/167 20130101; C12N 2710/10343
20130101; C12Q 1/6816 20130101; C12N 15/86 20130101; C12Q 1/6816
20130101 |
Class at
Publication: |
435/006 ;
435/456; 435/005 |
International
Class: |
C12Q 001/68; C12Q
001/70; C12N 015/861 |
Claims
What is claimed is:
1. A method of identifying a gene product, wherein the method
comprises: (a) providing a multiplicity of cells comprising a first
gene product; (b) introducing into the multiplicity of cells a
library of second nucleic acid sequences encoding second gene
products; (c) expressing the second nucleic acid sequences to
produce the second gene products such that the first gene product
and at least one of the second gene products contact; (d) causing
at least one complex to form between the first gene product, an
affinity molecule, and at least one of the second gene products;
(e) retrieving the complex; and (f) identifying at least one second
gene product of the complex.
2. The method of claim 1, wherein the first gene product is
produced in the multiplicity of cells via expression of a first
exogenous nucleic acid sequence encoding the first gene
product.
3. The method of claim 2, wherein the first nucleic acid sequence
is present in a plasmid.
4. The method of claim 2, wherein the first nucleic acid sequence
is present in a viral vector.
5. The method of claim 4, wherein the viral vector is an adenoviral
vector.
6. The method of claim 2, wherein the second nucleic acid sequence
is present in a plasmid.
7. The method of claim 2, wherein the second nucleic acid sequence
is present in a viral vector.
8. The method of claim 7, wherein the viral vector is an adenoviral
vector.
9. The method of claim 1, wherein identifying at least one second
gene product of the complex comprises dissociating the complex
between the first gene product, the affinity molecule, and at least
one of the second gene products such that the first gene product
and at least one of the second gene products remain intact.
10. The method of claim 1, wherein identifying at least one second
gene product of the complex comprises: (f1) producing charged
fragments from at least a portion of the complex; (f2) detecting
the charged fragments by a detector which produces a signal
corresponding to the mass-to-charge ratio of the charged fragments
and comprising information characteristic of at least one of the
second gene products; and (f3) evaluating the signal to identify at
least one of the second gene products.
11. The method of claim 1, wherein the affinity molecule is fixed
to a solid support.
12. The method of claim 11, wherein the solid support is a bead or
an affinity column.
13. The method of claim 11, wherein identifying at least one second
gene product of the complex comprises dissociating the complex
between the first gene product, the affinity molecule, and at least
one of the second gene products such that the first gene product
and at least one of the second gene products remain intact.
14. The method of claim 11, wherein identifying at least one second
gene product of the complex comprises: (f1) producing charged
fragments from at least a portion of the complex; (f2) detecting
the charged fragments by a detector which produces a signal
corresponding to the mass-to-charge ratio of the charged fragments
and comprising information characteristic of at least one of the
second gene products; and (f3) evaluating the signal to identify at
least one of the second gene products.
15. The method of claim 1, further comprising (g) identifying the
second nucleic acid sequence encoding at least one of the second
gene products.
16. The method of claim 1, wherein the first gene product and at
least one of the second gene products contact intracellularly.
17. The method of claim 1, wherein the first gene product and/or at
least one of the second gene products are secreted from the
multiplicity of cells and the first gene product and at least one
of the second gene products contact extracellularly.
18. The method of claim 1, wherein the first gene product comprises
a heterologous portion which associates with an affinity
molecule.
19. A method of identifying a gene product, wherein the method
comprises: (a) providing a viral vector comprising a first nucleic
acid sequence encoding a first gene product; (b) providing a
library of viral vectors, wherein each viral vector of the library
comprises a second nucleic acid sequence encoding a second gene
product; (c) transducing a multiplicity of host cells with the
viral vector comprising the first nucleic acid sequence and the
library of viral vectors comprising second nucleic acid sequences,
wherein the host cells are permissive for expression of the first
nucleic acid sequence and the second nucleic acid sequences and
production of the first gene product and the second gene products;
(d) expressing the first nucleic acid sequence and the second
nucleic acid sequences such that the first gene product and at
least one of the second gene products contact; (e) causing at least
one complex to form between the first gene product, an affinity
molecule, and at least one of the second gene products; (f)
retrieving the formed complex; and (g) identifying at least one
second gene product of the complex.
20. The method of claim 19, wherein the first gene product and at
least one of the second gene products contact intracellularly.
21. The method of claim 19, wherein the first gene product and/or
at least one of the second gene products are secreted from the
multiplicity of cells and the first gene product and at least one
of the second gene products contact extracellularly.
22. The method of claim 19, wherein the first gene product
comprises a heterologous portion which associates with an affinity
molecule.
23. The method of claim 19, wherein the viral vectors are
adenoviral vectors.
24. The method of claim 19, wherein identifying at least one second
gene product of the complex comprises: (g1) producing charged
fragments from at least a portion of the complex; (g2) detecting
the charged fragments by a detector which produces a signal
corresponding to the mass-to-charge ratio of the charged fragments
and comprising information characteristic of at least one of the
second gene products; and (g3) evaluating the signal to identify at
least one of the second gene products.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] This invention pertains to the use of genetic libraries to
identify a gene product of interest.
BACKGROUND OF THE INVENTION
[0002] The DNA and protein sciences have made great strides over
the past two decades. Researchers have accomplished the previously
unthinkable by sequencing the entire genomes of several
microorganisms. The genomes of several higher eukaryotes, including
mammals, are nearly completely sequenced and available on a variety
of databases. Potential use of the sequence information collected
to date is limitless if links between genetic sequence and cell
function can be established. In order to capitalize on the
seemingly endless supply of sequenced genomes, researchers have
developed genetic libraries that can be screened to associate a
nucleic acid sequence with a protein or peptide or cellular
function. In many instances, detection involves hybridizing to the
unknown DNA sequence a probe specific for a desired sequence. Yet,
such probes only detect sequence motifs, and peptide function
cannot be accurately predicted by the mere presence of motifs.
Alternatively, nucleic acid sequences are incorporated into a
vector and introduced into a host cell. The gene product encoded by
the nucleic acid is expressed and detected. Often, screening is
accomplished in vitro (see, for example, DeGraaf et al., Gene, 128
(1), 13-17 (1993)). For instance, nucleic acids from a library are
expressed and the peptides are collected and assayed. Yet, in vitro
assays are not predictive of in vivo activity, and the data
collected is not easily converted into information useful to, for
example, the pharmaceutical industry.
[0003] Despite the construction of genetic libraries, much of the
genome remains a mystery as to the function of encoded gene
products. Genomics data does not take into account pre- and
post-translational processing of gene products, nor does it give
any indication as the amount of peptide produced or whether a
peptide is active. Therefore, it would be advantageous and more
relevant to study the vast array of proteins within a cell. The
term "proteomics" has been used to refer to the large-scale
analysis of proteins and functional genomics.
[0004] Traditionally, the tool used for proteomics research is
two-dimensional polyacrylamide gels. Two-dimensional gel
electrophoresis allows the separation of many proteins from a cell
lysate based on charge and mass. Proteins separated in this manner
can be quantitated, catalogued, and analyzed. However,
two-dimensional gels are frequently not reproducible, and the
identification of the proteins separated on the gel is not
straightforward. In addition, only abundantly produced proteins can
be detected, as proteins are difficult to amplify. In addition,
some protein complexes, such as membrane protein complexes, are
hard to separate. Moreover, two-dimensional gel electrophoresis is
time-consuming and labor-intensive.
[0005] Like two-dimensional gel electrophoresis, yeast two-hybrid
systems also are useful in protein research. Yeast two-hybrid
systems are particularly useful in determining protein-protein
interactions. However, the yeast two-hybrid system has been plagued
with problems with false-negative and false-positive results and
usually takes months to develop even preliminary results.
[0006] Similarly, phage display libraries are used to express and
screen proteins for binding to a target molecule. In phage display
libraries, peptides of interest are expressed in the phage coat and
displayed to the environment. Phage display libraries have been
used to screen proteins in vitro by association of the expressed
peptide with a target ligand. However, the utility of phage display
libraries to associate function with a genetic sequence in vitro is
limited in that few targets have been identified, much less
successfully expressed in their native conformation. Phage display
libraries also have been utilized to identify peptides in vivo
(see, for example, U.S. Pat. No. 5,622,699 (Ruoslahti et al.)) Yet,
gene products identified by function in the context of phage may
not necessarily have similar function or activity in other contexts
or environments. For example, phage have limited utility in
screening in vitro and in vivo for ligands that are efficiently
internalized within a cell.
[0007] Protein arrays, similar to the DNA arrays commonly used in
genomics research, are currently available for the study of protein
interactions. Proteins are spotted on a metal chip, which can be
exposed to cell lysates, plasma, or targets from pharmaceutical
companies, to identify protein interactions. Yet, the fixation of
proteins on a surface can cause unfolding of the protein and
changes in active site conformations. In addition, the assays must
take place in vitro. Thus, the results observed using a chip assay
are not necessarily indicative of interactions that occur in
vivo.
[0008] Accordingly, there remains a need to provide a method of
screening genetic libraries. In particular, there remains a need in
the art for a method of screening the products of nucleic acid
sequences of a genetic library in their natural environment, e.g.,
intracellularly, to identify a gene product of interest. The
present invention provides a rapid, reliable, low-cost method for
observing gene product interactions and, advantageously, for
characterizing or identifying the encoded gene product. These and
other advantages of the present invention, as well as additional
inventive features, will be apparent from the description of the
invention provided herein.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides a method of identifying a
gene product. The method comprises providing a multiplicity of
cells comprising a first gene product. A library of second nucleic
acid sequences encoding second gene products is introduced into the
multiplicity of cells. The second nucleic acid sequences are
expressed in the multiplicity of cells to produce the second gene
products such that the first gene product and at least one of the
second gene products contact. Preferably, the first gene product
and at least one of the second gene products contact
intracellularly. The method further comprises causing a complex to
form between the first gene product, an affinity molecule, and at
least one of the second gene products, and subsequently retrieving
the complex. At least one second gene product of the complex then
is identified. Preferably, the first gene product is produced in
the multiplicity of cells via expression of a first exogenous
nucleic acid sequence encoding the first gene product.
[0010] The present inventive method further provides a method of
identifying a gene product comprising providing a viral vector
comprising a first nucleic acid sequence encoding a first gene
product and providing a library of viral vectors. Each member of
the library of viral vectors comprises a second nucleic acid
sequence encoding a second gene product. The method further
comprises transducing a multiplicity of host cells with the viral
vector comprising the first nucleic acid sequence and the library
of viral vectors comprising second nucleic acid sequences. The host
cells are permissive for expression of the first and second nucleic
acid sequences and production of the first and second gene
products. The first nucleic acid sequence and second nucleic acid
sequences are expressed such that the first gene product and the
second gene products contact. Preferably, the first gene product
and at least one of the second gene products contact
intracellularly. At least one complex is then caused to form
between the first gene product, an affinity molecule, and at least
one of the second gene products. The formed complex is retrieved,
and at least one second gene product of the complex is
identified.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The present invention provides a method of identifying a
gene product. The method comprises providing a multiplicity of
cells comprising a first gene product. Preferably, the first gene
product is produced in the multiplicity of cells via expression of
a first exogenous nucleic acid sequence encoding the first gene
product. A library of second nucleic acid sequences encoding second
gene products is introduced into the multiplicity of cells. The
method further comprises expressing the second nucleic acid
sequences to produce the second gene products such that the first
gene product and at least one of the second gene products contact.
Preferably, the first gene product and at least one of the second
gene products contact intracellularly. At least one complex is
caused to form between the first gene product, an affinity
molecule, and at least one of the second gene products. The method
further comprises retrieving the complex and identifying at least
one second gene product of the complex. The present inventive
method can be utilized to isolate and/or identify a gene product of
interest based on binding to another gene product, as well as
characterize gene product interactions.
[0012] In the present inventive method, a multiplicity of cells is
provided which comprises a first gene product. The first gene
product can be introduced into the multiplicity of cells using any
suitable method. For example, the first gene product can comprise a
signal sequence that allows passage of the first gene product
through the host cell membrane. Desirably, the first gene product
is produced in the multiplicity of cells via expression of a first
exogenous nucleic acid sequence encoding the first gene product.
Preferably, the cells are permissive for expression of the first
and second nucleic acid sequences and facilitate the production of
the first and second gene products. The multiplicity of cells can
be of any cell type, e.g., prokaryotic or eukaryotic, although
eukaryotic cells are preferred. Desirably, the multiplicity of
cells is the native environment for the first and/or the second
gene product. For example, if one of the gene products encodes an
enzyme active in the liver, preferably the multiplicity of cells is
derived from hepatocytes. The transduced multiplicity of cells can
be cultured in vitro or can be part of, e.g., in a tissue of, a
living organism, particularly a plant or animal, preferably a
mammal.
[0013] The first exogenous nucleic acid sequence and second nucleic
acid sequences can be part of any suitable entity that comprises a
nucleic acid (i.e., RNA or DNA) that encodes an RNA, protein, or a
polypeptide and which is capable of insertion into a multiplicity
of cells. Examples of suitable nucleic acid sequences include (1) a
DNA consisting, or consisting essentially, of a promoter and an RNA
coding region, (2) plasmids, including linear, circular, and
supercoiled plasmids, (3) cosmids, and (4) viral gene transfer
vectors. Examples of suitable gene transfer vectors comprising RNA
include (1) unencapsidated viral RNA, (2) heteronuclear RNA, (3)
messenger RNA, and (4) viral RNA that is encapsidated by one or
more coat proteins. The nucleic acid sequence optionally can be
associated with a liposome, cationic lipids, calcium ions, lithium
ions, an antigen binding protein, or any other agent that
facilitates the transfer of nucleic acids into a cell. By
"exogenous" is meant that the nucleic acid sequence (a) is not
native to the host cell or (b) is native to the host cell but is
located in a non-native position. For example, an exogenous nucleic
acid sequence can be native to a particular host cell, but is
expressed from a gene transfer vector, such as a viral vector.
[0014] Preferably, the nucleic acid sequence is present in a viral
vector. A viral vector useful in the context of the present
invention can be any viral vector that mediates insertion of the
nucleic acid sequence into a multiplicity of cells. Viral vectors
can comprise single-stranded ribonucleic acid (RNA),
double-stranded RNA, single-stranded deoxyribonucleic acid (DNA),
or double-stranded DNA. Examples of suitable viral vectors include,
but are not limited to, viral vectors derived, at least in part,
from adeno-associated virus (AAV)-based vectors, retroviral
vectors, herpes simplex virus (HSV)-based vectors, AAV-adenoviral
chimeric vectors, and adenovirus-based vectors. Any of these
expression vectors can be prepared using standard recombinant DNA
techniques described in, e.g., Sambrook et al., Molecular Cloning,
a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y. (1989), and Ausubel et al., Current Protocols
in Molecular Biology, Greene Publishing Associates and John Wiley
& Sons, New York, N.Y. (1994). The first nucleic acid sequence
and at least one second nucleic acid sequence of the library of
second nucleic acid sequences can be present in the same viral
vector. The use of viral vectors comprising the first nucleic acid
sequence or second nucleic acid sequences solves a problem
associated with current proteomics techniques. Previously, only
abundantly produced proteins were analyzed in that scarce proteins
were not detectable. However, the use of viral vectors, in
particular adenoviral vectors, enables the researcher to produce
sufficient quantities of previously limited gene products for
analysis.
[0015] In a preferred embodiment, the first exogenous nucleic acid
sequence is present in an adenoviral vector. Also desirably, the
second nucleic acid sequences making up the library of second
nucleic acid sequences are part of an adenoviral vector, thereby
generating a library of adenoviral vectors. Adenovirus is easy to
use, can be produced in high titers (i.e., up to about 10.sup.13
viral particles/ml), and transfers genes efficiently to
nonreplicating, as well as replicating cells (see, for example,
review by Crystal, Science, 270, 404-410 (1995)). Adenoviral
vectors exhibit a broad range of host- and cell-type specificity
and, if desired, can be manipulated to target a specific cell type.
Therefore, it is possible to identify a gene product based upon
gene product interactions specific to a cell type or tissue, which
is advantageous in instances where interaction of the first gene
product and the second gene product is dependent on cell-specific
post-translational modifications. In addition, adenoviral vectors
can be manipulated to accept large DNA molecules up to about 36 kb.
The characterization of the interaction between gene products or
the identity of a gene product can be determined in the context of
its natural intracellular or cell surface environment in vivo using
adenovirus to express the gene product(s). It will be appreciated
that identifying eukaryotic gene products and determining
eukaryotic protein-protein interactions in eukaryotic cells is more
accurate than other methods wherein eukaryotic gene products are
screened in bacterial cells or in soluble form in vitro.
[0016] Indeed, the use of viral vectors, in particular adenoviral
vectors, enables the identification of a second gene product that
interacts with a first gene product and characterization of the
gene product interactions in vivo. An entire library of second
nucleic acid sequences or a subset of the library can be
administered to an animal or introduced into host cells in vitro or
in vivo. Alternatively, the sublibrary comprising a population of
identical second nucleic acid sequences, a library comprising a
complexity of 1, can be introduced into an individual animal. The
library of second nucleic acid sequences can be administered to a
healthy animal, a diseased animal, or a transgenic animal in order
to screen the library in a particular in vivo environment.
[0017] In the context of the present invention, the adenoviral
vector can be derived from any serotype of adenovirus. Adenoviral
stocks that can be employed as a source of adenovirus can be
amplified from the adenoviral serotypes 1 through 51, which are
currently available from the American Type Culture Collection
(ATCC, Manassis, Va.), or from any other serotype of adenovirus
available from any other source. Preferably, the adenoviral vector
is derived from adenovirus serotypes 2 or 5. Preferred methods of
constructing and/or purifying adenoviral vectors are set forth in,
for example, U.S. Pat. No. 5,965,358 and International Patent
Applications WO 98/56937, WO 99/15686, and WO 99/54441.
[0018] The adenoviral vector is preferably deficient in at least
one gene function required for viral replication, thereby resulting
in a "replication-deficient" adenoviral vector. Preferably, the
adenoviral vector is deficient in at least one essential gene
function of the E1 region, e.g., the E1a region and/or the E1b
region, of the adenoviral genome. In addition to a deficiency in
the E1 region, the recombinant adenovirus also can have a mutation
in the major late promoter (MLP), as discussed in International
Patent Application WO 00/00628. More preferably, the vector is
deficient in at least one essential gene function of the E1 region
and at least part of the E3 region (e.g., an Xba I deletion of the
E3 region). Preferably, the adenoviral vector is "multiply
deficient," meaning that the adenoviral vector is deficient in one
or more essential gene functions required for viral replication in
each of two or more regions. For example, the aforementioned
E1-deficient or E1-, E3-deficient adenoviral vectors can be further
deficient in at least one essential gene of the E4 region and/or at
least one essential gene of the E2 region (e.g., the E2A region).
Adenoviral vectors deleted of the entire E4 region can elicit lower
host immune responses. Alternatively, the adenoviral vector can
lack all adenoviral sequences except the inverted terminal repeats
(ITRs) and packaging signal or ITRs and at least one adenoviral
promoter, thereby forming an adenoviral amplicon. Suitable
replication-deficient adenoviral vectors are disclosed in U.S. Pat.
Nos. 5,851,806 and 5,994,106 and International Patent Applications
WO 95/34671 and WO 97/21826.
[0019] Similarly, the coat protein of a viral vector, preferably an
adenoviral vector, can be manipulated to alter the binding
specificity or recognition of a virus for a viral receptor on a
potential host cell or to aid the vector in evading the immune
system. For adenovirus, such manipulations can include deletion of
regions of the fiber, penton, or hexon, insertions of various
native or non-native ligands into portions of the coat protein, and
the like. Manipulation of the coat protein can broaden the range of
cells infected by a viral vector or enable targeting of a viral
vector to a specific cell type. One direct result of this increased
efficiency of entry is that the virus, preferably, the adenovirus,
can bind to and enter numerous cell types which a virus comprising
wild-type coat protein typically cannot enter or can enter with
only a low efficiency. Alternatively, a chimeric virus coat protein
not selective for a specific type of eukaryotic cell can be
generated. In this embodiment, the chimeric virus coat protein
efficiently binds to a broader range of eukaryotic cells than a
wild-type virus coat, such as described in International Patent
Application WO 97/20051. Suitable modifications to a viral vector,
specifically an adenoviral vector, including modifications to coat
proteins, are described in U.S. Pat. Nos. 5,559,099; 5,731,190;
5,712,136; 5,770,442; 5,846,782; 5,926,311; 5,965,541; 6,057,155;
6,127,525; 6,153,435 and International Patent Applications WO
96/07734, WO 96/26281, WO 97/20051, WO 98/07865, WO 98/07877, WO
98/54346, and WO 00/15823.
[0020] By "library of second nucleic acid sequences" is meant a
collection of nucleic acid molecules, which are the same, i.e., the
nucleic acid sequences encode the same peptide or functional
nucleic acid sequence or variations thereof, or different, i.e.,
the nucleic acid sequences encode different peptides or functional
nucleic acid sequences. By "functional nucleic acid sequence" is
meant a nucleic acid sequence, i.e., DNA or RNA, that performs a
function or has an activity within a cell. An example of a
functional nucleic acid is antisense RNA that impedes transcription
or translation of a DNA or RNA sequence. The library of second
nucleic acid sequences can be obtained from any source. For
example, the second nucleic acid sequences can be genomic DNA
obtained from a source in nature that has not been genetically
modified. The library of second nucleic acid sequences also can be
obtained from an organism that has been modified to exhibit a
particular phenotype. The second nucleic acid sequences can
comprise cDNA or can be synthetically made using routine methods
known in the art. If required, the second nucleic acid sequences
can comprise pieces of larger molecules of DNA fragmented by
chemical, enzymatic, or mechanical means. The library of second
nucleic acid sequences also can comprise polymerase chain reaction
(PCR) products of DNA segments, and the like. Preferably, the
second nucleic acid sequences are obtained from a population of DNA
comprising a multiplicity of genetic elements.
[0021] The probability of identifying a gene product with a desired
activity depends greatly on the diversity of the genetic library.
It is, therefore, advantageous to mutate the DNA fragments to
obtain optimal diversity in the library of second nucleic acid
sequences. Nucleic acid sequences can be mutated using numerous
methods well understood in the art, such as, for example, exposure
to mutating chemical agents, e.g., ethidium bromide, recursive
shuffling (see, for example, International Patent Application WO
98/13485), error-prone PCR, error-prone transcription, and the
like. However, mutation of the second nucleic acid sequences is not
required. When screening genomic libraries to associate a function
with a nucleotide sequence, for example, the second nucleic acid
sequences are preferably not mutated.
[0022] The exogenous first nucleic acid sequence and the library of
second nucleic acid sequences can be introduced or inserted into
the multiplicity of host cells by any suitable method. Suitable
methods comprise infection (e.g., mediated by a coat-protein),
precipitation and co-incubation of the vector with suitable salts
(e.g., CaCl.sub.2 or LiCl), electroporation, particle bombardment,
needle-mediated direct injection, transduction, and any other
suitable methods for introducing nucleic acids into host cells.
[0023] The first nucleic acid sequence and/or the second nucleic
acid sequences can be constitutively expressed at a suitable level,
can be repressible, and/or can be induced in response to a stimulus
initiated from inside or outside the transduced host cells. If the
nucleic acid sequence requires transcription, any suitable promoter
can be used to drive the transcription of the nucleic acid
sequence. Suitable promoters comprise both viral and cellular
promoters. Suitable viral promoters are known in the art and
include, for instance, cytomegalovirus (CMV) promoters, such as the
CMV immediate-early promoter, promoters derived from human
immunodeficiency virus (HIV), such as the HIV long terminal repeat
promoter, Rous sarcoma virus (RSV) promoters, such as the RSV long
terminal repeat, mouse mammary tumor virus (MMTV) promoters, HSV
promoters, such as the herpes thymidine kinase promoter (Wagner et
al., Proc. Natl. Acad. Sci. (USA), 78, 144-145 (1981)), promoters
derived from SV40 or Epstein Barr virus, and the like. Preferably,
the viral promoter is an adenoviral promoter, such as the Ad2 or
Ad5 major late promoter and tripartite leader, a CMV promoter, or
an RSV promoter. Other suitable promoters for use in the methods of
the present invention include the regulatory sequences of the
metallothionine gene (Brinster et al., Nature, 296, 39-42 (1982)),
promoter elements from yeast or other fungi such as the Gal 4
promoter, the alcohol dehydrogenase promoter, the phosphoglycerol
kinase promoter, and the alkaline phosphatase promoter. Similarly,
promoters isolated from the genome of mammalian cells, such as the
.quadrature.-actin promoter or the muscle-creatine promoter, can be
employed. Induction or de-repression of transcription, where
appropriate, can be regulated by any suitable condition inside the
cell (such as, for example, adenosine diphosphate concentration,
stage of the cell cycle, and isoforms of p53 present) or by any
suitable external stimulus (such as, for example, hypoxia,
provision of metal ions to the cell, elevation of cellular
temperature, provision of a small signaling molecule, e.g., a
steroid, saccharide, or lipid, or exposure to non-background
radiation).
[0024] In certain embodiments, it can be advantageous to modulate
expression of the first and/or second nucleic acid sequences. A
suitable method of modulating expression of a nucleic acid sequence
comprises addition of site-specific recombination sites to the
expression vector. Contacting an expression vector comprising
site-specific recombination sites with a recombinase will either
up- or down-regulate transcription of a coding sequence, or
simultaneously up-regulate transcription one coding sequence and
down-regulate transcription of another, through the recombination
event. Use of site-specific recombination to modulate transcription
of a nucleic acid sequence is described, for example, U.S. Pat.
Nos. 5,801,030 and 6,063,627 and International Patent Application
WO 97/09439.
[0025] The first gene product and/or the second gene product(s) are
preferably RNA transcripts or a translated proteins, but also can
be post-transcriptional or post-translation products encoded by the
exogenous nucleic acid sequence and modified by enzymes or chemical
moieties in the cytosol, extracellular medium, or in a reaction
vessel. An advantage of the present inventive method is that gene
products to be screened can be modified intracellularly so as to
obtain a more accurate picture of the function of the encoded gene
products in vivo. The second nucleic acid sequences are expressed
to produce the second gene products such that the first gene
product and at least one of the second gene products contact (i.e.,
the second gene product(s) and the first gene product are expressed
under conditions wherein the second gene product(s) and first gene
product contact and, if appropriate, associate or bind. Preferably,
the first gene product and at least one of the second gene products
contact intracellularly. However, the first gene product and/or at
least one of the second gene products can be secreted gene
products, e.g., a secreted growth factor. Therefore, it is also
appropriate that the first gene product and at least one of the
second gene products contact extracellularly. Desirably, the gene
products directly or indirectly associate and form a complex
comprising the first gene product and at least one second gene
product. The association can be through any suitable manner, e.g.,
a covalent bond, a non-covalent bond, or both. Preferably, the bond
is stable and strong enough to allow for one or more washing or
isolation steps without causing or allowing the termination of the
association between the first and second gene products. The present
inventive method is superior to previously described proteomics
methods in that the first gene product and the second gene products
interact in the presence of intracellular molecules that can
interfere with recognition and binding. In other words, preferably
the gene products are allowed to contact prior to purifying the
gene products in order to achieve a more accurate interaction than
achieved with previous techniques. Previous methods of screening a
library in vitro cannot adequately mimic intracellular conditions,
unlike the method of the present invention.
[0026] The present inventive method further comprises causing at
least one complex to form between the first gene product, an
affinity molecule, and at least one of the second gene products,
and retrieving the formed complex. In the context of the present
invention, an affinity molecule is a molecule that associates with
the first gene product and can be used to at least partially
separate the first gene product (and molecules associated
therewith) from other molecules that do not associate with the
first gene product. The contacting of the first gene product with
the affinity molecule causes a complex to form between the affinity
molecule, the first gene product, and one or more second gene
products. Although forming an association between the first gene
product and an affinity molecule aids in isolation of the complex
of the first gene product and the second gene product(s), use of an
affinity molecule is not necessary so long as the complex can be
retrieved by other means. Exemplary of suitable affinity molecules
are antibodies or antigenically-reactive fragments thereof, metal
ions (e.g., zinc, cobalt, nickel, or copper), which are bound with
high-affinity by polyhistidine, as well as other DNAs, RNAs, and
proteins used in the art to separate molecules. Antibodies to the
first gene product can be generated using routine immunology
methods. Alternatively, the first gene product can comprise a
heterologous portion that associates with an affinity molecule.
Suitable heterologous portions, or tags, include the FLAG epitope,
the hemagluttenin (HA) epitope, or other antigenic epitopes
recognized by an antibody and that can be fused to the first gene
product.
[0027] Complexing the gene products with an affinity molecule
allows the partial or total separation of the first gene product
along with any associated second gene products, from other
molecules which can contact the gene products, such as those in the
cell, cell lysate, or (especially in the case of secreted proteins)
the cellular medium. The complex can be partially isolated and
retrieved by taking advantage of any suitable property of the
affinity molecule or the complex comprising the affinity molecule.
To facilitate retrieval of the complexes, preferably the affinity
molecule is fixed to (e.g., adhered to or forms part of) a solid
support, such as a bead or affinity column. The complex can be
separated, for instance, by affinity chromatography (such as
protein A or gel Blue A chromatography), molecular sieving
chromatography, selective adherence to a solid substrate, or
selective precipitation. Preferably, the complex is partially
isolated by selective precipitation or selective adherence to a
derivatized mass spectrometry slide.
[0028] Once the complex is retrieved, the second gene product can
be identified. Desirably, the complex between the first gene
product, the affinity molecule, and at least one of the second gene
products is dissociated such that the first gene product and at
least one second gene product remain intact. In other words, in
identifying the second gene product, preferably the affinity
molecule is removed from the complex. By keeping the binding pair
intact, the molecular weight of the associated gene products can be
determined. Alternatively, the complex can be completely
dissociated such that the affinity molecule, the first gene
product, and at least one second gene product are separated,
although this is less desired in some embodiments. At least one
second gene product then can be identified using a variety of
techniques. For example, the second gene product can be sequenced
using, for example, Edman degradation and subsequently compared to
amino acid sequences listed in various computer databases. If
desired, the second nucleic acid sequence (i.e., the nucleic acid
encoding the second gene product) can be identified.
[0029] Alternatively, the second gene product can be identified
based on physical properties. In one embodiment, identifying at
least one second gene product of the complex comprises producing
charged fragments from at least a portion of the complex. In this
aspect, the present inventive method is superior to other methods
in proteomics in that separation of the first gene product and
second gene product(s) is not required. It is, of course,
preferable to avoid additional steps (e.g., selective
precipitation, liquid chromatography (other than desalting), gel
electrophoresis, and differential centrifugation) because these
steps increase the complexity of the method and increase time and
cost requirements. The charged fragments are detected by a detector
which produces a signal corresponding to the mass-to-charge ratio
of the charged fragments. Optionally, the complex can be isolated
and disassociated into its component molecules prior to charging
and fragmenting, although this is less preferred in some
embodiments. Additionally, individual molecular components of the
complex can be re-isolated from the disassociated complex before
producing charged fragments such that the resulting sample signal
will be significantly simplified. The signal comprises information
characteristic of at least the second gene product and, optionally,
the first gene product and/or affinity molecule. The produced
signal is then evaluated to identify the second gene product.
Optionally, the signal comprising information characteristic of the
second gene product is compared to a standard signal generated from
the first gene product alone or a complex of the first gene product
and the affinity molecule.
[0030] The generated signal desirably comprises a fingerprint that
allows the identification of the second gene product, or at least
its sequence. If the complex is not dissociated, at least one
second gene product can be detected by the presence of data in the
signal that does not reflect mass-to-charge values corresponding to
charged fragments of the first gene transfer vector product or the
affinity molecule. Any suitable technique can be used to
characterize the second gene product once it is detected. For
example, the second gene product can be characterized by comparison
with a standard signal, or by other techniques such as, but not
limited to, subjecting the second gene product to chromatography,
differential gradient analysis (e.g., a cesium chloride gradient
column or sucrose gradient), electrophoresis, centrifugation,
ELISA, or any combination of the aforementioned or other
techniques.
[0031] In one particular aspect of the invention, the
characterization of at least one detected second gene product can
be achieved by subjecting one or more charged fragments of the
second gene product to further fragmentation and analysis. In this
particular aspect, secondary charged fragments are produced from a
charged fragment(s) generated from the second gene product. The
secondary charged fragments then can be detected by a detector that
produces a signal corresponding to the mass-to-charge ratio of the
detected secondary second gene product fragment. This signal (e.g.,
a spectrum) corresponding to the mass-to-charge ratio of the
secondary second gene product charged fragments allows for rapid
identification of the second gene product.
[0032] Any suitable technique or combination of techniques can be
used to produce charged fragments from at least one second gene
product, detect the charged gene product fragments, and generate a
signal corresponding to the mass-to-charge ratio of the charged
second gene product fragments. The gene products are charged (e.g.,
a proton is added to an electrically neutral gene product without
cleaving the gene product) and, preferably, charged and fragmented.
The charged fragments can be produced by any suitable technique,
for example, a technique comprising contacting at least one
retrieved second gene product with light, energy, or a chemical.
Preferably, the charged fragments will be produced by contacting at
least one second gene product with radiation, electrons, or
protons, and more preferably with protons. Any suitable form of
radiation, electrons, or protons can be used in the present
inventive method to produce charged fragments from the second gene
product. "Radiation" in the context of the present invention refers
to any emission of energy in the form of electromagnetic waves,
acoustic waves, or particles. Examples of radiation suitable in the
context of the present invention include, but are not limited to,
radio waves, microwaves, visible light, ultraviolet (UV) light, far
UV and infrared rays, x-rays, gamma-rays, infrasonic waves, sonic
waves, ultrasonic waves, and .alpha.- and .beta.-rays of
radioactivity. The electrons can be in any suitable form. For
example, the electrons can be in the form of electron beams or
individual electrons. Similarly, the protons can be in any suitable
form. Moreover, the radiation, electrons, and protons can be from
any suitable source. For example, radiation can be emitted from a
natural source (e.g., radioactive cobalt), an x-ray device, or a
laser. Preferably, the charged fragment of the second gene product
is further fragmented, for example, by collision-induced
dissociation (CID), to produce the secondary charged fragments of
at least one second gene product.
[0033] The radiation, electrons, and protons can have any suitable
characteristics. In particular, the radiation can have any suitable
wavelength, for example, in the infrared spectrum of the
ultraviolet spectrum. Similarly, any pulse width suitable to
produce charged fragments can be utilized in the present inventive
method. For example, the pulse generated by a laser can have a
width of from about 1 to about 10 nanoseconds (e.g., 3 nanoseconds)
and can have a width up to about 20, 50, 75, 100, 200, or even
5,000 nanoseconds. Moreover, variable pulse widths and multiple
repeated pulses can be used to produce the charged fragments.
[0034] The charged fragments can be in any state or quantity
suitable for fragment detection. For example, a sample comprising
the gene transfer vector product can be vaporized at the time the
charged fragments are produced, such that the charged fragments
enter into a gaseous or vapor state, have greater mobility, and are
subject to easier detection. The charged fragments can have any
suitable velocity at the time of production that allows fragments
to be accelerated, after they are produced, to a desired velocity
before the charged fragments are detected. The charged fragments
can pass through a field-free region, wherein the velocity of the
charged fragments within the field-free region is proportional to
the mass-to-charge ratio of the charged fragments. One example of
such a field-free region is an electric field, particularly an
electric field wherein lighter charged fragments have a higher
velocity than heavier charged fragments. While acceleration of
charged fragments is not required, if the charged fragments are
accelerated, any level of acceleration sufficient for detection of
the charged fragments can be used. For example, the charged
fragments can be accelerated to a fixed kinetic energy by
contacting the charged fragments with an electric potential.
[0035] The detection of the charged fragments of at least one
second gene product is accomplished by any suitable technique. The
detection can include, for example, measuring the time-of-flight of
the detected charged fragments, wherein the time-of-flight is the
approximate time required for a charged fragment to travel a
distance across a field-free region. A signal corresponding to the
time-of-flight then can be generated.
[0036] While the fragmentation and detection steps of the present
inventive method can be performed in any suitable manner, they are
preferably performed by the use of an analytical device, most
preferably by the use of an ion source and a mass analyzer, such as
are present in mass spectrometers. Suitable ion sources comprise
electron impact, fast ion or atom bombardment, ion spray, field
desorption, laser desorption (including, but not limited to
matrix-assisted laser desorption ionization (MALDI)), plasma
desorption, thermospray, electrospray ionization (including, but
not limited to, nanoelectrospray ionization and/or capillary
electrospray), inductively coupled plasma, chemical ionization
(including, but not limited to atmospheric pressure chemical
ionization (APCI)), and atmospheric pressure ionization (including,
but not limited to, APCI). Preferred ion sources comprise MALDI,
thermospray, electrospray ionization (ESI), and atmospheric
pressure ionization (API).
[0037] Suitable mass analyzers comprise quadrupole analyzers (e.g.,
single-quadrupole or triple quadrupole), ion trap or quistor
analyzers, time-of-flight (TOF) analyzers, TOF/TOF analyzers,
hybrid quadrupole/time-of-flight mass analyzers, magnetic and
electromagnetic analyzers, ion cyclotron resonance analyzers, and
Fourier transform mass analyzers. Preferably, the charged fragments
are detected by a mass analyzer selected from the group of mass
analyzers consisting of a time-of-flight, a single quadrupole, a
triple quadrupole, a hybrid quadrupole/time-of-flight, a Fourier
transform, an ion trap, a single focusing magnetic deflection
instrument, and a double focusing magnetic focusing instrument.
Other mass spectrometers that can be used in the context of the
present invention comprise an electrospray ionization mass
spectrometer, a plasma desorption spectrometer, a thermospray
ionization spectrometer, and a laser desorption mass
spectrometer.
[0038] Most preferably, a MALDI-TOF spectrometer with precursor
selector (or "ion gate") capabilities, a quadrapole electrospray
spectrometer, or any spectrometer capable of focusing on one or
more signals that correspond to at least one second gene product is
used to produce, detect, and analyze the originally produced and,
where applicable, secondary charged second gene product fragments.
A MALDI-TOF spectrometer with precursor selector capabilities is
particularly preferred because it is capable of accurately
detecting (and focusing) on a signal corresponding to at least one
second gene product, isolating the originally-produced second gene
product fragment, and rapidly performing further analysis on the
isolated second gene product fragment.
[0039] Improved resolution and faster analysis can sometimes be
obtained using tandem mass spectrometry. Additionally, by using
tandem mass spectrometry (e.g., by using a spectrometer possessing
two detectors and a reflectron), identification of monoisotopic
masses versus average masses is possible.
[0040] The charged fragments can have any suitable size for
detection, sample signal production, and analysis. For example, the
produced and detected charged fragments can have molecular weights
of up to 5,000 daltons (i.e., atomic mass units), 30,000 daltons,
150,000 daltons, or even 300,000 daltons or more. Similarly, the
charged fragments can have any suitable mass-to-charge ratio for
detection, sample signal production and analysis. For example, the
produced and detected charged fragments can have mass-to-charge
ratios (m/z) of at least 50, 100, 200, 400, 500, 1,000, 2,500,
5,000, 10,000, 20,000, or even 25,000, 300,000 or more.
[0041] The sample signal generated upon detection of the charged
fragments can be any type of signal that allows for evaluation and,
optionally, comparison to a standard signal, and can be generated
in any suitable manner. Similarly, a standard signal can be any
signal, in any form, that allows for useful comparison with a
sample signal to detect, for example, protein-protein interactions.
The standard signal can be a single signal or a group (or series)
of signals. The sample and standard signals can be associated with
any suitable single mass-to-charge ratios. The sample signal and
standard signal can be presented in similar or different (though
preferably similar) formats, measurements, or units. For example, a
suitable standard signal can be a signal that is produced from
techniques similar to those that are used to generate the sample
signal. More specifically, the standard signal can be a signal that
is generated from a standard source, e.g., the first gene
product.
[0042] An example of this aspect of the invention is the
identification of the amino acid composition of a second gene
product by the technique described above. After identifying a
signal corresponding to at least one second gene product, the
second gene product can be further fragmented (e.g., by CID). By
controlling fragmentation, charged fragments corresponding to
individual constituent amino acids can be produced. Signals
corresponding to the amino acids then are generated by a detector,
allowing rapid determination of the amino acid composition of the
polypeptide.
[0043] Any of the individual steps of the present inventive method
can be repeated two or more times. For example, the production of
charged fragments and the detection of the charged fragments can be
repeated two or more times to provide a satisfactory sample
signal(s). Methods of producing and detecting charged fragments to
produce a signal corresponding to the mass-to-charge ratio of the
charged fragments to evaluate genetic materials are further
described in U.S. Pat. No. 5,965,358 and International Patent
Applications WO 00/12765 and WO 01/11087.
[0044] Therefore, in one embodiment of the present invention, the
method of identifying a gene product comprises providing a viral
vector comprising a first nucleic acid sequence encoding a first
gene product and providing a library of viral vectors, wherein each
viral vector of the library comprises a second nucleic acid
sequence encoding a second gene product. Desirably, the viral
vectors are adenoviral vectors. The method further comprises
transducing a multiplicity of host cells with the viral vector
comprising the first nucleic acid sequence and the library of viral
vectors comprising second nucleic acid sequences using any suitable
method, e.g., routine methods known in the art. Preferably, the
viral vectors are adenoviral vectors, as described herein. The host
cells are permissive for expression of the first and second nucleic
acid sequences and production of the first and second gene
products. The first nucleic acid sequence and the second nucleic
acid sequences are then expressed in the multiplicity of host cells
such that the first gene product and at least one of the second
gene products contact. Preferably, the first gene product and at
least one of the second gene products contact intracellularly.
Alternatively, the first gene product and/or at least one of the
second gene products is secreted from the multiplicity of cells,
and the first gene product and at least one of the second gene
products contact extracellularly. To aid in identifying at least
one second gene product, at least one complex is caused to form
between the first gene product, an affinity molecule, and at least
one of the second gene products. At least one complex then is
retrieved, and at least one second gene product of the complex is
identified. Preferably, the first gene product comprises a
heterologous portion which associates with the affinity molecule to
ensure specific binding of the affinity molecule to the first gene
product.
[0045] While at least one second gene product can be identified
using a variety of methods, preferably identifying at least one
second gene product of the complex comprises producing charged
fragments from at least a portion of the complex. The charged
fragments are detected by a detector which produces a signal
corresponding to the mass-to-charge ratio of the charged fragments
and comprising information characteristic of the second gene
product. The signal is then evaluated to identify the second gene
product, as described herein.
[0046] A library of viral vectors, e.g., adenoviral vectors,
preferably comprises, consists essentially of, or consists of a
multiplicity of viral vectors comprising a multiplicity of genetic
elements. Any number of individual viral vectors can make up the
library of viral vectors. Likewise, any number of second nucleic
acid sequences can make up a library of second nucleic acid
sequences. The complexity of the library of viral vectors can vary
according to the particular embodiment. By "complexity" is meant
the number of unique individuals in the library. Preferably, the
complexity of the library of viral vectors is about 1 to about
10.sup.11 colony forming units. More preferably, the complexity of
the library is about 1 to about 10.sup.6 colony forming units, or
unique individuals. In other words, the complexity of the library
viral vectors preferably is 2 (e.g., 3, 5, 10, 10.sup.2, 10.sup.3,
etc.) to 10.sup.9 (e.g., 10, 10.sup.2, 10.sup.3, 10.sup.4,
10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, or any other integer within
the range of 2 to 109) particles.
[0047] The present inventive method can be performed multiple times
in order to identify or isolate a gene product. For example, the
present inventive method can be performed at least 2, 3, 4, or more
times (e.g., at least 5, 7, 10, 15, or 20 times) in order to
identify a gene product of interest. Moreover, the identified gene
products can be re-entered into the present inventive method to
identify additional gene products. In other words, a previously
identified second gene product can be used in subsequent rounds of
screening as the "first gene product" in order to identify gene
products that interact with that previously identified second gene
product. Using the identified gene products in this manner, and
repeating the process any number of times as described above,
entire cellular pathways can be mapped.
[0048] The binding of at least one of the second gene products to
the first gene product can be verified or evaluated by exposing the
first gene product and at least one second gene product to an
entity which specifically inhibits association of the first and
second gene products (e.g., a binding inhibitor). For example, the
first gene product and at least one of the second gene products are
peptides, and the second gene product(s) is identified. To confirm
the identity of the second gene product(s) or to evaluate the
peptide-peptide interactions between the first and second gene
products, the first gene product and the identified second gene
product(s) are allowed to contact in the presence of, for example,
a small molecule or biomolecule known to interfere with binding.
The peptide-peptide interaction can be evaluated, for instance, to
confirm that the second peptide specifically binds the first
peptide. In addition, the affinity of the second gene product for
the first gene product can be characterized. For example, if using
mass spectroscopy, the relative intensity of the peaks of the
spectra corresponding to the binding pair can be used to evaluate
the affinity of the binding partners.
[0049] In this regard, the techniques described herein can be used
to evaluate peptide-peptide interactions, as well as identifying
gene products. For instance, once interacting gene products are
identified, the system described herein can be used to identify
targets, i.e., small molecules, that inhibit, disrupt, augment, or
do not impact the peptide-peptide interaction. In this aspect, the
first peptide and the second peptide are allowed to contact in the
presence of a potential target, e.g., a small molecule, compound,
or biomolecule that enhances, inhibits, or has no effect on binding
of the first and second peptides. Any peptide-peptide interactions
are evaluated by any suitable method. Peptide-peptide interactions
are preferably evaluated by producing charged fragments from at
least a portion of the complex, detecting the charged fragments
using a detector that produces a signal corresponding to the
mass-to-charge ratio, and evaluating the signal to evaluate the
peptide-peptide interaction. The signal generated from the
peptide-peptide complex that formed in the presence of the target
is compared to a standard signal generated from a peptide-peptide
complex formed without the target. Comparison of the signals
provides information regarding the inhibitory or enhancing effect
of the target on peptide-peptide binding. Any of the methods
described herein can be repeated any number of times to screen a
library of potential targets, such as those generated in
pharmaceutical research. The second gene product used to screen
target molecules as described above can be produced by expressing a
second nucleic acid sequence selected from a library comprising a
complexity of one.
[0050] The present inventive method also can be utilized to
construct personal, patient-specific therapeutics. It is apparent
in the clinic that potential therapeutics do not act similarly in
all patients. It is, therefore, desirable to create therapeutic
agents and treatment regimens tailored for individual patients. To
determine the efficacy of a potential therapeutic, a library of
second nucleic acid sequences can be constructed from genomic DNA
from an individual patient, tissue, or organ. A first nucleic acid
sequence is provided that encodes the potential therapeutic agent.
The first nucleic acid sequence and the second nucleic acid
sequence(s) are expressed under conditions wherein the first gene
product and at least one second gene product interact to form a
complex. Formation of a complex is indicative of an interaction of
the therapeutic agent with a cellular factor encoded by the second
nucleic acid sequence and, therefore, provides valuable information
to a clinician as to the likelihood that a candidate biotherapeutic
would affect a biological response. Moreover, the second gene
product can be identified to determine what cellular factor
associates with the potential therapeutic. Alternatively, instead
of constructing a genetic library from a patient's cells, a
population of cells (such as cells isolated from a biopsy) can be
lysed to provide a library of cellular factors into which is
introduced the potential therapeutic in vitro. Also alternatively,
a population of cells (e.g., a library of cells) is infected with
adenoviral vectors comprising the first nucleic acid sequence. Any
complexes formed by the therapeutic and the cellular factor(s) are
isolated and the cellular factor identified using, for example,
mass spectroscopy, as described herein. Using the techniques
described herein, relevant protein-protein and protein-therapeutic
interactions in specific tissues can be elucidated.
[0051] In addition, the data generated from the techniques
described herein, including data concerning the identification of
the gene products, the interaction of gene products, and the
screening of target molecules which do or do not affect the
interaction of gene products, can be compiled, organized, screened,
and/or sorted in any fashion. For example, the data generated using
any of the techniques described herein can be compiled into a
database, e.g., a database accessible via the internet, to provide
access to the gathered information in a meaningful format.
[0052] The following example further illustrates the present
invention but, of course, should not be construed as in any way
limiting its scope.
EXAMPLE
[0053] This example illustrates the association of a first gene
product, the identity of which is known in advance, with a second
gene product, and the subsequent detection of the second gene
product.
[0054] A cDNA ("a first exogenous nucleic acid sequence") encoding
a chimeric fusion protein of the 14.7K gene product and a
heterologous region encoding a polypeptide that associates with
high affinity to antibodies specific for hemagluttenin (HA) ("a
first gene product"), and separately a cDNA ("a second nucleic acid
sequence") encoding the gene product of the adenoviral-E3 14.7K
gene ("a second gene product"), were subcloned by standard
techniques into a DNA to form expression cassettes that were
transcriptionally regulated by a CMV immediate-early promoter and
an SV40 early polyadenylation signal. Each of these two expression
cassettes was inserted into the deleted E1 region of separate E1-,
E3-deleted adenoviral gene transfer vectors. The resulting
adenoviral gene transfer vectors were named Ad14.7HA and Ad14.7,
respectively. A stock of each adenoviral gene transfer vector was
produced by standard techniques.
[0055] HEK-293 cells were co-infected with a sample of each of the
Ad14.7HA stock and the Ad14.7 stock. About 17 hours later (i.e.,
after infection), the 293 cells were lysed with detergents, and the
insoluble fraction was removed by low-speed centrifugation. A
monoclonal anti-HA antibody ("an affinity molecule") bound by
Protein A-sepharose beads (PAS-beads, Boehringer Mannheim) then was
added to the soluble fraction of the cell lysate. The mixture was
incubated with rocking at 4.degree. C. overnight.
[0056] A complex comprising the 14.7K-HA protein, the 14.7K
protein, the monoclonal antibody, and the PAS-beads (i.e., a
complex of the first gene product, the affinity molecule, and the
second gene product) formed, which complex was separated from the
soluble fraction of the lysate by centrifugation. The complex was
washed three times with phosphate buffered saline. The complex was
suspended in 1% trifluoroacetic acid (TFA), which caused the
complex to disassociate. The PAS-beads, which are insoluble, were
allowed to settle, and the supernatant fraction was transferred to
a new container.
[0057] The supernatant fraction, in which proteins from the complex
were suspended, was dried and resuspended in 0.1% TFA, and applied
to a ZipTip C18 (Millipore) desalting column to reduce the
concentration of salts, detergents, and any other small molecules
which may have been associated with the complex. The proteins were
eluted from the ZipTip C18 desalting-column with a mixture
containing 50% acetonitrile, 0.1% TFA, and 10 mg/ml sinapinic
acid.
[0058] Charged fragments of the eluted material were produced, and
the mass-to-charge ratios of the charged fragments were detected
with a MALDI-TOF mass spectrometer (PE BIO Systems). The sample
signal produced by the mass spectrometer comprised peaks with
mass-to-charge ratios equivalent to the predicted mass of the 14.7
and 14.7HA proteins. Evaluation of the sample signal indicated that
the adenoviral E3-14.7K protein (i.e., the second gene product)
associates with a chimeric fusion protein comprising the E3-14.7K
protein and an HA-epitope (i.e., the first gene product).
[0059] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0060] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0061] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Of course, variations of those preferred
embodiments will become apparent to those of ordinary skill in the
art upon reading the foregoing description. The inventors expect
skilled artisans to employ such variations as appropriate, and the
inventors intend for the invention to be practiced otherwise than
as specifically described herein. Accordingly, this invention
includes all modifications and equivalents of the subject matter
recited in the claims appended hereto as permitted by applicable
law. Moreover, any combination of the above-described elements in
all possible variations thereof is encompassed by the invention
unless otherwise indicated herein or otherwise clearly contradicted
by context.
* * * * *