U.S. patent application number 10/458720 was filed with the patent office on 2004-12-09 for methods for conducting assays for enzyme activity on protein microarrays.
This patent application is currently assigned to Protometrix, Inc.. Invention is credited to Schweitzer, Barry, Zhou, Fang X..
Application Number | 20040248323 10/458720 |
Document ID | / |
Family ID | 33490454 |
Filed Date | 2004-12-09 |
United States Patent
Application |
20040248323 |
Kind Code |
A1 |
Zhou, Fang X. ; et
al. |
December 9, 2004 |
Methods for conducting assays for enzyme activity on protein
microarrays
Abstract
The present invention relates to methods of conducting assays
for enzymatic activity on microarrays useful for the large-scale
study of protein function, screening assays, and high-throughput
analysis of enzymatic reactions. The invention relates to methods
of using protein chips to assay the presence, amount, activity
and/or function of enzymes present in a protein sample on a protein
chip. In particular, the methods of the invention relate to
conducting enzymatic assays using a microarray wherein a protein
and a substance are immobilized on the surface of a solid support
and wherein the protein and the substance are in proximity to each
other sufficient for the occurrence of an enzymatic reaction
between the substance and the protein. The invention also relates
to microarrays that have an enzyme and a substrate immobilized on
their surface wherein the enzyme and the substrate are in proximity
to each other sufficient for the occurrence of an enzymatic
reaction between the enzyme and the substrate.
Inventors: |
Zhou, Fang X.; (New Haven,
CT) ; Schweitzer, Barry; (Cheshire, CT) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
Protometrix, Inc.
|
Family ID: |
33490454 |
Appl. No.: |
10/458720 |
Filed: |
June 9, 2003 |
Current U.S.
Class: |
436/518 ;
435/23 |
Current CPC
Class: |
C12Q 1/00 20130101 |
Class at
Publication: |
436/518 ;
435/023 |
International
Class: |
C12Q 001/37; G01N
033/543 |
Claims
We claim:
1. A method for assaying an enzymatic reaction, the method
comprising: (a) incubating at least one protein and at least one
substance under conditions conducive to the occurrence of an
enzymatic reaction between the protein and the substance, wherein
(i) the protein and the substance are immobilized on the surface of
a solid support; (ii) the protein and the substance are in
proximity sufficient for the occurrence of said enzymatic reaction;
and (iii) the protein and the substance are not identical; and (b)
determining whether said enzymatic reaction occurs.
2. The method of claim 1, wherein the protein, the substance, or
the protein and the substance, are purified.
3. The method of claim 1, wherein the substance is a mixture of
different substances.
4. The method of claim 1, wherein the method comprises immobilizing
a protein and a substance on the surface of the solid support.
5. The method of claim 4, wherein the protein is immobilized prior
to immobilizing the substance.
6. The method of claim 4, wherein the substance is immobilized
prior to immobilizing the protein.
7. The method of claim 4, wherein the substance and the protein are
immobilized simultaneously.
8. The method of claim 1, wherein the substance is a known
substrate for the type of enzymatic activity assayed in said
enzymatic reaction, and said determining step determines whether
said protein is an enzyme having said type of enzymatic
activity.
9. The method of claim 1, wherein the protein is an enzyme known to
have the type of enzymatic activity assayed in said enzymatic
reaction, and said determining step determines whether said
substrate is a substrate for said type of enzymatic activity.
10. The method of claim 8, wherein the protein comprises a region
that is homologous to the catalytic domain of an enzyme that is
known to have the type of enzymatic activity assayed in said
enzymatic reaction.
11. The method of claim 9, wherein the enzyme is an oxidoreductase,
a transferase, a hydrolase, a lyase, an isomerase, or a ligase.
12. The method of claim 11, wherein the enzyme is a kinase.
13. The method of claim 1, wherein (i) the substance is a known
substrate for the type of enzymatic activity assayed in said
enzymatic reaction; and (ii) the protein is known to catalyze the
type of enzymatic activity assayed in said enzymatic reaction.
14. The method of claim 13, wherein said incubating step is done in
the presence of one or more test molecules so as to determine
whether said test molecules modulate said enzymatic reaction; and
said determining step comprises detecting whether a change in the
amount of said enzymatic reaction occurs relative to the amount of
said enzymatic reaction in the absence of the test molecules.
15. The method of claim 14, wherein said determining step comprises
detecting a decrease in the amount of said enzymatic reaction
relative to the amount of said enzymatic reaction in the absence of
the test molecules, thereby identifying the test molecules as an
inhibitor of said enzymatic reaction.
16. The method of claim 14, wherein said determining step comprises
detecting an increase in the amount of said enzymatic reaction
relative to the amount of said enzymatic reaction in the absence of
the test molecules, thereby identifying the test molecules as an
activator of said enzymatic reaction.
17. The method of claim 13, wherein the substance is known to be a
substrate of said enzyme.
18. The method of claim 1, wherein said at least one protein is one
of a plurality of different proteins organized on the surface of
the solid support in a positionally addressable array.
19. The method of claim 18, wherein the substance is coated onto
the surface of the solid support.
20. The method of claim 18, wherein each protein of the plurality
of proteins is in proximity with the substance sufficient for the
occurrence of an enzymatic reaction between the protein and the
substance, and said determining step comprises determining for at
least a portion of said plurality of proteins whether said
enzymatic reaction occurs.
21. The method of claim 1, wherein said at least one substance is
one of a plurality of different substances organized on the surface
of the solid support in a positionally addressable array.
22. The method of claim 21, wherein the protein is coated onto the
surface of the solid support.
23. The method of claim 21, wherein each substance of the plurality
of different substances is in proximity with the protein sufficient
for the occurrence of an enzymatic reaction between the protein and
the substance, and said determining step comprises determining for
at least a portion of said plurality of different substances
whether said enzymatic reaction occurs.
24. The method of claim 18, wherein said plurality consists of
between 2 different proteins and 100 different proteins.
25. The method of claim 18, wherein said plurality consists of
between 100 different proteins and 1,000 different proteins.
26. The method of claim 18, wherein said plurality consists of
between 1,000 different proteins and 10,000 different proteins.
27. The method of claim 21, wherein said plurality consists of
between 2 different substances and 100 different substances.
28. The method of claim 21, wherein said plurality consists of
between 100 different substances and 1,000 different
substances.
29. The method of claim 21, wherein said plurality consists of
between 1,000 different substances and 10,000 different
substances.
30. The method of claim 1, wherein (i) a first plurality of
different substances are organized on the surface of the solid
support in a positionally addressable array, and (ii) a second
plurality of different proteins are organized on the surface of the
solid support in a positionally addressable array.
31. The method of claim 30, wherein said first plurality consists
of between 2 different substances and 100 different substances, and
said second plurality consists of between 2 different proteins and
100 different proteins.
32. The method of claim 30, wherein said first plurality consists
of between 100 different substances and 1,000 different substances,
and said second plurality consists of between 100 different
proteins and 1,000 different proteins.
33. The method of claim 30, wherein said first plurality consists
of between 1,000 different substances and 10,000 different
substances, and said second plurality consists of between 100
different proteins and 10,000 different proteins.
34. The method of claim 1, wherein the substance, the protein, or
the substance and the protein, are covalently bound to the surface
of the solid support.
35. The method of claim 1, wherein the substance, the protein, or
the substance and the protein, are non-covalently immobilized to
the surface of the solid support.
36. The method of claim 1, wherein the substance, the protein, or
the substrate and the protein, are immobilized to the surface of
the solid support via a linker.
37. The method of claim 1, wherein the method further comprises
quantifying the enzymatic reaction.
38. The method of claim 1, wherein the solid support has at least
one well and wherein the well comprises one or more different
proteins immobilized on the surface of the solid support within the
well.
39. The method of claim 38, wherein the solid support has at least
two wells and wherein each well comprises the same one or more
different proteins immobilized on the surface of the solid support
within the well.
40. The method of claim 1, wherein the solid support has at least
one well and wherein each well comprises one or more different
substances immobilized on the surface of the solid support within
the well.
41. The method of claim 40, wherein the solid support has at least
two wells and wherein each well comprises the same set of one or
more different substances immobilized on the surface of the solid
support within the well.
42. The method of claim 1, wherein said determining step comprises
measuring a change in a detectable signal resulting from said
enzymatic reaction.
43. A positionally addressable array comprising at least one known
enzyme and at least one candidate substrate of the enzyme, wherein
(i) the enzyme and the substrate are immobilized on the surface of
a solid support; (ii) the enzyme and the substrate are in proximity
sufficient for the occurrence of the enzymatic reaction catalyzed
by the enzyme between the enzyme and the substrate; and (iii) the
enzyme and the substrate are not identical to each other.
44. A positionally addressable array comprising at least one known
substrate of an enzymatic reaction and at least one candidate
enzyme for the catalysis of the enzymatic reaction, wherein (i) the
enzyme and the substrate are immobilized on the surface of a solid
support; (ii) the enzyme and the substrate are in proximity
sufficient for the occurrence of the enzymatic reaction between the
enzyme and the substrate; and (iii) the enzyme and the substrate
are not identical to each other.
45. A positionally addressable array comprising at least one known
substrate of an enzymatic reaction and at least one enzyme that is
known to catalyze the enzymatic reaction, wherein (i) the enzyme
and the substrate are immobilized on the surface of a solid
support; (ii) the enzyme and the substrate are in proximity
sufficient for the occurrence of the enzymatic reaction between the
enzyme and the substrate; and (iii) the enzyme and the substrate
are not identical to each other.
46. The positionally addressable array of claim 43, 44, or 45,
further comprising a reaction mixture, said reaction mixture being
(i) in contact with the enzyme and the substrate, and (ii)
conducive to the occurrence of the enzymatic reaction.
47. The positionally addressable array of claim 43, 44, or 45,
wherein the enzyme, the substrate, or the enzyme and the substrate,
are purified.
48. The positionally addressable array of claim 43, 44, or 45,
wherein the surface of the solid support is coated with the enzyme
or with the substrate.
49. The positionally addressable array of claim 43, 44, or 45,
wherein said substrate is one of a plurality of different
substrates.
50. The positionally addressable array of claim 49, wherein each
different substrate is in proximity with an enzyme sufficient for
the occurrence of the enzymatic reaction between the substrate and
the enzyme.
51. The positionally addressable array of claim 43, 44, or 45,
wherein said enzyme is one of a plurality of different enzymes.
52. The positionally addressable array of claim 51, wherein each
different enzyme is in proximity with a substrate sufficient for
the occurrence of the enzymatic reaction between the substrate and
the enzyme.
53. The positionally addressable array of claim 43, 44, or 45,
wherein said enzyme is one of a plurality of different enzymes and
said substrate is one of a plurality of different substrates.
54. A positionally addressable array comprising at least one known
enzyme immobilized on the surface of a solid support, wherein the
surface is modified to allow for the immobilization of a candidate
substrate of the enzymatic reaction catalyzed by said enzyme in
proximity with the enzyme sufficient for the occurrence of the
enzymatic reaction between the enzyme and the substrate.
55. A positionally addressable array comprising at least one
substrate of an enzymatic reaction immobilized on the surface of a
solid support, wherein the surface is modified to allow for the
immobilization of a candidate enzyme capable of catalyzing said
enzymatic reaction in proximity with the substrate sufficient for
the occurrence of the enzymatic reaction between the enzyme and the
substrate.
56. A kit comprising the positionally addressable array of claim
43, 44, or 45, and a container comprising a reaction mixture
conducive to the occurrence of the enzymatic reaction.
Description
1. FIELD OF THE INVENTION
[0001] The present invention relates to methods of conducting
assays for enzymatic activity on microarrays useful for the
large-scale study of protein function, screening assays, and
high-throughput analysis of enzymatic reactions. The invention
relates to methods of using protein chips to assay the presence,
amount, activity and/or function of enzymes present in a protein
sample on a protein chip. In particular, the methods of the
invention relate to conducting enzymatic assays using a microarray
wherein a protein and a substance are immobilized on the surface of
a solid support and wherein the protein and the substance are in
proximity to each other sufficient for the occurrence of an
enzymatic reaction between the substance and the protein. The
invention also relates to microarrays that have an enzyme and a
substrate immobilized on their surface wherein the enzyme and the
substrate are in proximity to each other sufficient for the
occurrence of an enzymatic reaction between the enzyme and the
substrate.
2. BACKGROUND OF THE INVENTION
[0002] A daunting task in the post-genome sequencing era is to
understand the functions, modifications, and regulation of every
protein encoded by a genome (Fields et al., 1999, Proc Natl Acad.
Sci. 96:8825; Goffeau et al., 1996, Science 274:563). Currently,
much effort is devoted toward studying gene, and hence protein,
function by analyzing mRNA expression profiles, gene disruption
phenotypes, two-hybrid interactions, and protein subcellular
localization (Ross-Macdonald et al., 1999, Nature 402:413; DeRisi
et al., 1997, Science 278:680; Winzeler et al., 1999, Science
285:901; Uetz et al., 2000, Nature 403:623; Ito et al., 2000, Proc.
Natl. Acad. Sci. U.S.A. 97:1143). Important advances in this effort
have been possible, in part, by the ability to analyze thousands of
gene sequences in a single experiment using gene chip technology.
Although these studies are useful, transcriptional profiles do not
necessarily correlate well with cellular protein levels or protein
activities. Thus, the analysis of biochemical activities can
provide information about protein function that complements genomic
analyses to provide a more complete picture of the workings of a
cell (Zhu et al., 2001, Curr. Opin. Chem. Biol. 5:40; Martzen, et
al., 1999, Science 286:1153; Zhu et al., 2000, Nat. Genet. 26:283;
MacBeath, 2000, Science 289:1760; Caveman, 2000, J. Cell Sci.
113:3543).
[0003] Currently, biochemical analyses of protein function are
performed by individual investigators studying a single protein at
a time. This is a very time-consuming process since it can take
years to purify and identify a protein based on its biochemical
activity. The availability of an entire genome sequence makes it
possible to perform biochemical assays on every protein encoded by
the genome. Based on sequence comparison, genes encoding for
proteins with a particular enzymatic activity can be identified.
However, a detailed analysis of an individual proteins' biochemical
properties, such as, substrate specificity, kinetic profile and
sensitivities to inhibitors, is a time-consuming process. Thus,
high-throughput ways of analyzing the biochemical activities of
proteins are required.
[0004] It would be useful to analyze hundreds or thousands of
protein samples using a single protein chip. Such approaches lend
themselves well to high throughput experiments in which large
amounts of data can be generated and analyzed. Microtiter plates
containing 96 or 384 wells have been known in the field for many
years. However, the size (at least 12.8 cm.times.8.6 cm) of these
plates makes them unsuitable for the large-scale analysis of
proteins.
[0005] Recently devised methods for expressing large numbers of
proteins with potential utility for biochemical genomics in the
budding yeast Saccharomyces cerevisiae have been developed. ORFs
have been cloned into an expression vector that uses the GAL
promoter and fuses the protein to a polyhistidine (e.g., HISX6)
label. This method has thus far been used to prepare and confirm
expression of about 2000 yeast protein fusions (Heyman et al.,
1999, "Genome-scale cloning and expression of individual open
reading frames using topoisomerase I-mediated ligation," Genome
Res. 9:383-392). Using a recombination strategy, about 85% of the
yeast ORFs have been cloned in frame with a GST coding region in a
vector that contains the CUP I promoter (inducible by copper), thus
producing GST fusion proteins (Martzen et al., 1999, "A biochemical
genomics approach for identifying genes by the activity of their
products," Science 286:1153-1155). Martzen et al. used a pooling
strategy to screen the collection of fusion proteins for several
biochemical activities (e.g., phosphodiesterase and
Appr-1-P-processing activities) and identified the relevant genes
encoding these activities.
[0006] Several groups have recently described microarray formats
for the screening of protein activities (Zhu et al., 2000, Nat.
Genet. 26:283; MacBeath et al., 2000, Science 289:1763; Arenkov et
al, 2000, Anal. Biochem 278:123). In addition, a collection of
overexpression clones of yeast proteins have been prepared and
screened for biochemical activities (Martzen et al., 1999, Science
286: 1153).
[0007] Photolithographic techniques have been applied to making a
variety of arrays, from oligonucleotide arrays on flat surfaces
(Pease et al., 1994, "Light-generated oligonucleotide arrays for
rapid DNA sequence analysis," PNAS 91:5022-5026) to arrays of
channels (U.S. Pat. No. 5,843,767) to arrays of wells connected by
channels (Cohen et al., 1999, "A microchip-based enzyme assay for
protein kinase A," Anal Biochem. 273:89-97). Furthermore,
microfabrication and microlithography techniques are well known in
the semiconductor fabrication area. See, e.g., Moreau,
Semiconductor Lithography: Principals, Practices and Materials,
Plenum Press, 1988.
[0008] Screening a large number of proteins or even an entire
proteome would entail the systematic probing of biochemical
activities of proteins that are produced in a high throughput
fashion, and analyzing the functions of hundreds or thousands of
proteins samples in parallel (Zhu et al., 2000, Nat. Genet. 26:283;
MacBeath et al., 2000, Science 289:1763; Arenkov et al, 2000, Anal.
Biochem 278:123; International Patent Application publication WO
01/83827 and WO 02/092118). In vitro assays have previously been
conducted using random expression libraries or pooling strategies,
both of which have shortcomings (Martzen et al., 1999, Science
286:1153; Bussow et al., 2000, Genomics 65:1). Specifically, random
expression libraries are tedious to screen, and contain clones that
are often not full-length. Another recent approach has been to
generate defined arrays and screen the array using a pooling
strategy (Martzen et al. 1999, Science 286:1153). The pooling
strategy obscures the actual number of proteins screened, however,
and the strategy is cumbersome when large numbers of positives are
identified.
[0009] Therefore, there remains a need in the art for the
large-scale analysis of biochemical functions which would allow
assessing the activities, in a high-throughput manner, of a large
number of proteins.
[0010] Citation or identification of any reference in this
application shall not be considered as admission that such
reference is available as prior art to the present invention.
3. SUMMARY OF THE INVENTION
[0011] The present invention provides protein chips and methods
useful for the study of protein activities in a high-throughput
manner. The present invention also provides methods for identifying
substrates of enzymes and modulators of enzymatic activities. The
invention is directed to methods of using protein chips to assay
the presence, amount, functionality, activity and sensitivity to
modulators of enzymes. In particular, the invention is directed to
methods of conducting assays for enzymatic activity on protein
microarrays. In certain embodiments, a method of the invention for
assaying an enzymatic reaction comprises the following steps: (a)
incubating at least one protein and at least one substance under
conditions conducive to the occurrence of an enzymatic reaction
between the protein and the substance, wherein (i) the protein and
the substance are immobilized on the surface of a solid support;
(ii) the protein and the substance are in proximity sufficient for
the occurrence of said enzymatic reaction; and (iii) the protein
and the substance are not identical; and (b) determining whether
said enzymatic reaction occurs.
[0012] The methods of the invention can be used to determine
whether a protein catalyzes an enzymatic reaction of interest. In
this embodiment, the substance is a known substrate of the
enzymatic reaction to be tested, and substrate and protein are
incubated in a reaction mixture that provides conditions conducive
to the occurrence of the enzymatic reaction and that provides any
cofactors required by the enzymatic reaction.
[0013] The methods of the invention can be used to determine
whether a substance is a substrate of an enzymatic reaction of
interest. In this embodiment, the protein is an enzyme known to
catalyze the reaction of interest and substrate and protein are
incubated in a reaction mixture that provides conditions conducive
to the occurrence of the enzymatic reaction and that provides any
cofactors required by the enzymatic reaction.
[0014] The protein, the substance, or the protein and the substance
to be used with the methods of the invention can be purified. The
substance can be a known substrate for the type of enzymatic
activity assayed in the enzymatic reaction of a method of the
invention, and the determining step of a method of the invention
determines whether said protein is an enzyme having said type of
enzymatic activity. The protein can be an enzyme known to have the
type of enzymatic activity assayed in said enzymatic reaction, and
said determining step determines whether said substrate is a
substrate for said type of enzymatic activity. If the substance is
a known substrate for the type of enzymatic activity assayed in
said enzymatic reaction, the protein can comprise a region that is
homologous to the catalytic domain of an enzyme that is known to
have the type of enzymatic activity assayed in said enzymatic
reaction. In certain embodiments, the enzyme is an oxidoreductase,
a transferase, a hydrolase, a lyase, an isomerase, or a ligase. In
a more specific embodiment, the enzyme is a kinase.
[0015] In certain embodiments, the substance is a known substrate
for the type of enzymatic activity assayed in the enzymatic
reaction of a method of the invention; and the protein is known to
catalyze the type of enzymatic activity assayed in said enzymatic
reaction. In these embodiments, substrate and enzyme can be
incubated under conditions conducive to the occurrence the
enzymatic reaction in the presence of one or more test molecules so
as to determine whether said test molecules modulate said enzymatic
reaction; and said determining step comprises detecting whether a
change in the amount of said enzymatic reaction occurs relative to
the amount of said enzymatic reaction in the absence of the test
molecules. The determining step can comprise detecting a decrease
in the amount of said enzymatic reaction relative to the amount of
said enzymatic reaction in the absence of the test molecules,
thereby identifying the test molecules as an inhibitor of said
enzymatic reaction. In other embodiments, said determining step can
comprise detecting an increase in the amount of said enzymatic
reaction relative to the amount of said enzymatic reaction in the
absence of the test molecules, thereby identifying the test
molecules as an activator of said enzymatic reaction. In certain
embodiments, the substance is known to be a substrate of the
enzyme.
[0016] In certain embodiments, said at least one protein of a
method of the invention is one of a plurality of different proteins
organized on the surface of the solid support in a positionally
addressable array. The plurality of different proteins can consist
of between 2 different proteins and 100 different proteins. The
plurality of different proteins can consist of between 100
different proteins and 1,000 different proteins. The plurality of
different proteins can consist of between 1,000 different proteins
and 10,000 different proteins. The substance can be coated onto the
surface of the solid support. In certain, more specific
embodiments, each protein of the plurality of proteins is in
proximity with the substance sufficient for the occurrence of an
enzymatic reaction between the protein and the substance, and said
determining step comprises determining for at least a portion of
said plurality of proteins whether said enzymatic reaction
occurs.
[0017] In certain embodiments, said at least one substance of a
method of the invention is one of a plurality of different
substances organized on the surface of the solid support in a
positionally addressable array. The protein can be coated onto the
surface of the solid support. In certain, more specific
embodiments, each substance of the plurality of different
substances is in proximity with the protein sufficient for the
occurrence of an enzymatic reaction between the protein and the
substance, and said determining step comprises determining for at
least a portion of said plurality of different substances whether
said enzymatic reaction occurs. The plurality of different
substances can consist of between 2 different substances and 100
different substances. The plurality of different substances can
consist of between 100 different substances and 1,000 different
substances. The plurality of different substances can consist of
between 1,000 different substances and 10,000 different
substances.
[0018] In certain embodiments, a first plurality of different
substances is organized on the surface of the solid support in a
positionally addressable array, and a second plurality of different
proteins is organized on the surface of the solid support in a
positionally addressable array. Said first plurality can consists
of between 2 different substances and 100 different substances, and
said second plurality can consist of between 2 different proteins
and 100 different proteins. In other embodiments, the first
plurality consists of between 100 different substances and 1,000
different substances, and said second plurality consists of between
100 different proteins and 1,000 different proteins. In even other
embodiments, said first plurality consists of between 1,000
different substances and 10,000 different substances, and said
second plurality consists of between 100 different proteins and
10,000 different proteins. In certain embodiments, copies of said
first plurality are present 1 time, 2 times, 3, 4, 5, 6, 7, 8, 9,
10, 16, 24, at least 30, at least 50, or at least 100 times on the
surface of the solid support. In certain embodiments, copies of
said second plurality are present 1 time, 2 times, 3, 4, 5, 6, 7,
8, 9, 10, 16, 24, at least 30, at least 50, or at least 100 times
on the surface of the solid support.
[0019] In certain embodiments, the substance, the protein, or the
substance and the protein, are covalently bound to the surface of
the solid support. In other embodiments, the substance, the
protein, or the substance and the protein, are non-covalently
immobilized to the surface of the solid support. In even other
embodiments, the substance, the protein, or the substrate and the
protein, are immobilized to the surface of the solid support via a
linker.
[0020] In certain embodiments, a method of the invention also
comprises quantifying the enzymatic reaction. In certain
embodiments, the determining step of a method of the invention
further comprises measuring a change in a detectable signal
resulting from said enzymatic reaction.
[0021] In certain embodiments, the solid support has at least one
well and wherein the well comprises one or more different proteins
immobilized on the surface of the solid support within the well. In
certain embodiments, the solid support has at least two wells and
wherein each well comprises the same one or more different proteins
immobilized on the surface of the solid support within the
well.
[0022] In certain embodiments, the solid support has at least one
well and wherein each well comprises one or more different
substances immobilized on the surface of the solid support within
the well. The solid support can have at least two wells and wherein
each well comprises the same set of one or more different
substances immobilized on the surface of the solid support within
the well.
[0023] The invention provides, a positionally addressable array
comprising at least one known enzyme and at least one candidate
substrate of the enzyme, wherein (i) the enzyme and the substrate
are immobilized on the surface of a solid support; (ii) the enzyme
and the substrate are in proximity sufficient for the occurrence of
the enzymatic reaction catalyzed by the enzyme between the enzyme
and the substrate; and (iii) the enzyme and the substrate are not
identical to each other.
[0024] The invention also provides a positionally addressable array
comprising at least one known substrate of an enzymatic reaction
and at least one candidate enzyme for the catalysis of the
enzymatic reaction, wherein (i) the enzyme and the substrate are
immobilized on the surface of a solid support; (ii) the enzyme and
the substrate are in proximity sufficient for the occurrence of the
enzymatic reaction between the enzyme and the substrate; and (iii)
the enzyme and the substrate are not identical to each other.
[0025] The invention further provides a positionally addressable
array comprising at least one known substrate of an enzymatic
reaction and at least one enzyme that is known to catalyze the
enzymatic reaction, wherein (i) the enzyme and the substrate are
immobilized on the surface of a solid support; (ii) the enzyme and
the substrate are in proximity sufficient for the occurrence of the
enzymatic reaction between the enzyme and the substrate; and (iii)
the enzyme and the substrate are not identical to each other.
[0026] In certain embodiments, a positionally addressable array of
the invention further comprises a reaction mixture, said reaction
mixture being (i) in contact with the enzyme and the substrate, and
(ii) conducive to the occurrence of the enzymatic reaction.
[0027] In certain embodiments, the enzyme, the substrate, or the
enzyme and the substrate of a positionally addressable array of the
invention are purified. In certain embodiments, the surface of the
solid support of a positionally addressable array of the invention
is coated with the enzyme or with the substrate.
[0028] In certain embodiments, the coating of an array with
substance or with protein covers the entire array. In other
embodiments, the coating of an array with substance or with protein
covers a part of the array. In certain embodiments, the area
covered by the coating is larger than the area covered by a protein
or substrate that is printed on the surface of the array.
[0029] In certain embodiments, a substrate on a positionally
addressable array of the invention is one of a plurality of
different substrates. In certain embodiments, each different
substrate is in proximity with an enzyme sufficient for the
occurrence of the enzymatic reaction between the substrate and the
enzyme.
[0030] In certain embodiments, an enzyme on a positionally
addressable array of the invention is one of a plurality of
different enzymes. In certain embodiments, each different enzyme is
in proximity with a substrate sufficient for the occurrence of the
enzymatic reaction between the substrate and the enzyme.
[0031] In certain embodiments, an enzyme on a positionally
addressable array of the invention is one of a plurality of
different enzymes and a substrate on a positionally addressable
array of the invention is one of a plurality of different
substrates.
[0032] The invention also provides a positionally addressable array
comprising at least one known enzyme immobilized on the surface of
a solid support, wherein the surface is modified to allow for the
immobilization of a candidate substrate of the enzymatic reaction
catalyzed by said enzyme in proximity with the enzyme sufficient
for the occurrence of the enzymatic reaction between the enzyme and
the substrate.
[0033] The invention further provides a positionally addressable
array comprising at least one substrate of an enzymatic reaction
immobilized on the surface of a solid support, wherein the surface
is modified to allow for the immobilization of a candidate enzyme
capable of catalyzing said enzymatic reaction, wherein the enzyme
is immobilized in proximity with the substrate sufficient for the
occurrence of the enzymatic reaction between the enzyme and the
substrate.
[0034] The invention also provides a kit comprising the
positionally addressable array of the invention, and a container
comprising a reaction mixture conducive to the occurrence of the
enzymatic reaction.
[0035] In certain embodiments, the present invention is directed to
protein chips, which are positionally addressable arrays comprising
a plurality of proteins, with each protein being immobilized at a
different position on a solid support and wherein at least one
substrate of an enzymatic reaction is also immobilized to the solid
support such that a protein and a substrate are in proximity
sufficient for the occurrence of the enzymatic reaction between the
protein and the substrate. In certain specific embodiments, the
plurality of proteins represents a substantial proportion of all
proteins expressed in a single species, wherein translation
products of one open reading frame are considered a single
protein.
[0036] In certain embodiments, the present invention provides a
positionally addressable array comprising a plurality of proteins,
with each protein being immobilized at a different position on a
solid support, wherein the plurality of proteins comprises at least
1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, or 99% of all proteins expressed in a single species and
wherein at least one substrate of an enzymatic reaction is also
immobilized to the solid support such that a protein and a
substrate are in proximity sufficient for the occurrence of the
enzymatic reaction between the protein and the substrate. In a
specific embodiment, protein isoforms and splice variants are
counted as a single protein.
[0037] In another embodiment, the present invention provides a
positionally addressable array comprising a plurality of proteins,
with each protein being immobilized at a different position on a
solid support, wherein the plurality of proteins comprises at least
1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 500, 1000, 1500, 2000,
2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 100, 000,
500,000 or 1,000,000 protein(s) and wherein at least one substrates
of an enzymatic reaction is also immobilized to the solid support
such that a protein and a substrate are in proximity sufficient for
the occurrence of the enzymatic reaction between the protein and
the substrate.
[0038] In another embodiment, the invention provides a positionally
addressable array comprising a plurality of proteins, with each
protein being immobilized at a different position on a solid
support, wherein the plurality of proteins in aggregate comprise
proteins encoded by at least 1000 different known genes of a single
species and wherein at least one substrate of an enzymatic reaction
is also immobilized to the solid support such that a protein and a
substrate are in proximity sufficient for the occurrence of the
enzymatic reaction between the protein and the substrate.
[0039] In a further embodiment, the proteins are organized on the
array according to a classification of proteins. The classification
can be by abundance, function, functional class, enzymatic
activity, homology, protein family, association with a particular
metabolic or signal transduction pathway, association with a
related metabolic or signal transduction pathway, or
posttranslational modification. In a specific embodiment, the
invention provides a positionally addressable array comprising a
plurality of proteins with a specific enzymatic activity wherein at
least one substrate of the enzymatic reaction that is catalyzed by
the enzymatic activity of the proteins on the array is also
immobilized on the solid support and wherein a protein and a
substrate are in proximity sufficient for the occurrence of the
enzymatic reaction between the protein and the substrate.
[0040] In certain embodiments, the invention provides a
positionally addressable array of proteins on a solid support
wherein at least one substrate of an enzymatic reaction is
immobilized on the solid support, wherein the solid support
comprises glass, ceramics, nitrocellulose, amorphous silicon
carbide, castable oxides, polyimides, polymethylmethacrylates,
polystyrenes, gold or silicone elastomers.
[0041] In one embodiment, the surface of the solid support is a
flat surface, such as, but not limited to, glass slides. Dense
protein arrays can be produced on, for example, glass slides, such
that chemical reactions and assays can be conducted, thus allowing
large-scale parallel analysis of the presence, amount, and/or
functionality of proteins. In a specific embodiment, the flat
surface array has proteins bound to its surface via a
3-glycidooxypropyltrimethoxysilane (GPTS) linker.
[0042] In certain embodiments, the invention relates to microarrays
and methods of using the microarrays for enzymatic assays, wherein
an enzyme is immobilized to the surface of a solid support and
wherein a plurality of different substrates is also immobilized to
the surface of the solid support such that enzyme and substrates
are in physical contact. Each individual substrate of the plurality
of substrates is immobilized at a different position of the surface
of the solid support.
[0043] In certain embodiments, the amount of enzyme needed in an
enzymatic reaction performed using the methods of the present
invention to obtain a detectable signal is at least 10-fold, at
least 100-fold, at least 1000-fold or at least 10,000-fold lower
compared to performing the assay wherein only the enzyme or only
the substrate is immobilized.
[0044] 4. Definitions and Abbreviations
[0045] As used in this application, "protein" refers to a peptide
or polypeptide. Proteins can be prepared from recombinant
overexpression in an organism, preferably bacteria, yeast, insect
cells or mammalian cells, or produced via fragmentation of larger
proteins, or chemically synthesized.
[0046] As used in this application, "enzyme" refers to any protein
with a catalytic activity.
[0047] As used in this application, "functional domain" is a domain
of a protein which is necessary and sufficient to give a desired
functional activity. Examples of functional domains include, inter
alia, domains which exhibit an enzymatic activity such as
oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase
activity. In more specific embodiments, a functional domain
exhibits kinase, protease, phosphatase, glycosidase, or acetylase
activity. Other examples of functional domains include those
domains which exhibit binding activity towards DNA, RNA, protein,
hormone, ligand or antigen.
[0048] Each protein or substrate of an enzymatic reaction on a chip
is preferably located at a known, predetermined position on the
solid support such that the identity of each protein or probe can
be determined from its position on the solid support. Further, the
proteins and probes form a positionally addressable array on a
solid support.
[0049] As used herein, the term "purified" refers to a molecule, a
substance or a protein that is substantially free of different
molecules of the same type, substances of the same type, or
proteins, respectively, that are associated with it in its original
state (from which it is purified). Preferably, a molecule is at
least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%,
99.98%, 99.998%, 99.9998%, 99.99998% or at least 99.999998% free of
such different molecules, wherein, if the molecule is in solution,
the solvent is not a different molecule. Preferably, a substance is
at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%,
99.9%, 99.98%, 99.998%, 99.9998%, 99.99998% or at least 99.999998%
free of such different substances. Preferably, a protein is at
least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%,
99.98%, 99.998%, 99.9998%, 99.99998% or at least 99.999998% free of
such different proteins.
1 ABBREVIATIONS Abbreviation RIE Reactive Ion Etching GST
glutathione-S-transferase GPTS 3-glycidooxypropyltrimethoxysilane
ORF Open reading frame FRET Fluorescence Resonance Energy
Transfer
4.1. BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1. Autoradiographs of kinase reactions on two different
microarrays. The top microarray was coated with substrate and the
bottom array was without substrate coating.
[0051] FIG. 2. Autoradiographs of kinase reactions on different
microarrays demonstrating the superior signal-to-noise ratio if the
slides are treated with an aldehyde. The aldehyde-treated slides
were obtained from TeleChem International, Inc. The slide shown as
FAST is a nitrocellulose coated slide (Schleicher & Schuell).
The slide shown as GAPS is coated with an amino-silane surface
(Corning.RTM.).
[0052] FIG. 3. Inhibitor Specificity Profiling. Kinase reactions
with 50 different human kinases .alpha.-axis) were performed in the
presence of 100 .mu.M H89 inhibitor (diamond squares), 100 .mu.M
rottlerin inhibitor (squares) or 100 .mu.M PP2 inhibitor
(triangles). The percentages of inhibition are plotted on the
y-axis for each inhibitor.
[0053] FIG. 4a. Dose-response curve for inhibition of the kinases
FYN (squares), EPHB3 (diamond squares) and PRKCD (triangles) by
PP2. PP2 concentration is shown on the x-axis and percent kinase
activity compared to the control reaction without inhibitor is
shown on the y-axis.
[0054] FIG. 4b. Dose-response curve for inhibition of the kinases
FYN (squares), EPHB3 (diamond squares) and PRKCD (triangles) by
Staurosporine. Staurosponrin concentration is shown on the x-axis
and percent kinase activity compared to the control reaction
without inhibitor is shown on the y-axis.
[0055] FIG. 5. Shows the susceptibility of different human kinases
to different inhibitors. Eight different inhibitors were tested on
a plurality of different human kinases on one slide.
5. DETAILED DESCRIPTION OF THE INVENTION
[0056] The invention is directed to methods of conducting assays
for enzymatic activity on protein microarrays. In the methods of
the invention, a substance and a protein, both immobilized on the
surface of the microarray, are in proximity with each other
sufficient for the occurrence of an enzymatic reaction between the
substance and the protein. The present invention also provides
methods of using protein chips to assay the presence, amount,
functionality, activity and sensitivity to modulators of enzymes.
The invention further provides microarrays containing a substance
and a protein, both immobilized on the surface of the microarray,
wherein the substance and the protein are in proximity with each
other sufficient for the occurrence of an enzymatic reaction
between the substance and the protein, for use, e.g., to determine
if the substance is a substrate and/or if the protein is an enzyme
that acts on the substrate, for the enzymatic activity being
assayed, or to identify inhibitors of the enzymatic reaction.
[0057] In certain embodiments, the methods of the invention can be
used to identify enzymes that catalyze a specific reaction. In
certain embodiments, the methods of the invention can be used to
identify enzymes that use a specific substrate. In these
embodiments, one or more proteins that are candidates for the
enzyme that catalyzes the reaction of interest are immobilized on a
protein chip for use with the invention.
[0058] In certain embodiments, the methods of the invention can be
used to identify substrates of an enzymatic activity of interest.
In certain embodiments, the methods of the invention can be used to
identify substrates that are used by enzymes having a specific
catalytic activity. In certain embodiments, the methods of the
invention can be used to identify substrates that are used by a
class of enzymes or by a specific enzyme of interest. In these
embodiments, one or more substances that are candidates for
substrates of the enzymatic activity of interest are immobilized on
the surface of a solid support.
[0059] In certain embodiments, the methods of the invention can be
used to identify modulators of enzyme activity. In such screening
assays, a molecule that increases or decreases the enzymatic
activity being assayed can be identified. In certain embodiments,
molecules that alter the substrate specificity of an enzyme can be
identified. In other embodiments, the kinetic properties of an
inhibitor, an activator or a molecule that alters the substrate
specificity of an enzyme can be assessed.
[0060] In certain embodiments, a method of the invention for
assaying an enzymatic reaction comprises the following steps: (a)
incubating at least one protein and at least one substance under
conditions conducive to the occurrence of an enzymatic reaction
between the protein and the substance, wherein (i) the protein and
the substance are immobilized on the surface of a solid support;
(ii) the protein and the substance are in proximity sufficient for
the occurrence of said enzymatic reaction; and (iii) the protein
and the substance are not identical; and (b) determining whether
said enzymatic reaction occurs.
[0061] In certain embodiments, a method of the invention comprises
the steps of (i) immobilizing a substance on a solid support; (ii)
printing a plurality of different proteins on the solid support
such that a substance and a protein are in proximity sufficient for
the occurrence of said enzymatic reaction between the substance and
the proteins; and (iii) detecting the occurrence of the enzymatic
reaction. In certain embodiments, a method of the invention
comprises the steps of (i) immobilizing a protein on a solid
support; (ii) printing a plurality of different substances on the
solid support such that a substance and a protein are in proximity
sufficient for the occurrence of said enzymatic reaction; and (iii)
detecting the occurrence of the enzymatic reaction between the
substance and the protein. In certain, more specific embodiments,
the occurrence of the enzymatic reaction is visualized and/or
quantified by a detectable signal.
[0062] In certain embodiments, the plurality of proteins is printed
on the surface of the solid support in a positionally addressable
fashion such that the identity of a protein that is located at a
specific position of the array can be easily determined. In certain
embodiments, the plurality of substances is printed on the surface
of the solid support in a positionally addressable fashion such
that the identity of a substance that is located at a particular
position of the array can be easily determined. A positionally
addressable array provides a configuration such that each substance
and/or protein of interest is located at a known, predetermined
position on the solid support such that the identity of each
substrate and/or protein can be determined from its position on the
array.
[0063] In certain embodiments, the surface of the solid support is
coated with a substrate of an enzymatic reaction and the plurality
of different proteins is printed on top of the substrate coating.
In certain, more specific embodiments, each protein of the
plurality of proteins is immobilized at a different position of the
surface of the solid support. In other embodiments, the surface of
the solid support is coated with a plurality of different
substrates and the plurality of different proteins is printed on
top of each substrate. In certain, more specific embodiments, the
different substrates are coated on the surface as a mixture. In
other embodiments, each substrate of the plurality of substrates is
coated in a different area of the solid support. In other
embodiments, a substrate is printed on the surface of the solid
support and the plurality of different proteins is printed on top
of the substrate. In certain embodiments, a plurality of different
substrates is printed on the surface of the solid support and the
plurality of different proteins is printed on top of the
substrates. In a specific embodiment, all possible
substrate-protein combinations of a set of proteins of interest and
a set of substrates of interest are present on a single microarray.
In certain, more specific, embodiments, the substrates and/or the
proteins are purified.
[0064] In certain embodiments, the different proteins of the
plurality of different proteins are immobilized at different
positions on the surface of the solid support. In certain, more
specific embodiments, at least one protein of the plurality of
different proteins is immobilized at at least 2, 3, 4, 5, 6, 7, 8,
9, 10, 12, 15, 20, 25, 50, or at at least 100 different locations
on the surface of the solid support. In a preferred embodiment,
each protein is immobilized at at least 4 different positions on
the surface of the solid support.
[0065] In certain embodiments, the different substrates of the
plurality of different substrates are immobilized at different
positions on the surface of the solid support. In certain, more
specific embodiments, at least one substrate of the plurality of
different substrates is immobilized at at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 12, 15, 20, 25, 50, or at at least 100 different
locations on the surface of the solid support. In a preferred
embodiment, each substrate is immobilized at at least 4 different
positions on the surface of the solid support.
[0066] In certain embodiments, the surface of the solid support is
coated with an enzyme and a plurality of different substances is
printed on top of the enzyme coating. In certain, more specific
embodiments, each substance of the plurality of substances is
immobilized at a different position of the surface of the solid
support. In other embodiments, the surface of the solid support is
coated with a plurality of different enzymes and a plurality of
different substances is printed on top of each different enzyme. In
certain, more specific embodiments, the different enzymes are
immobilized on the surface of the solid support as a mixture. In
other, more specific embodiments, the different enzymes are
immobilized in different regions of the surface of the solid
support. In other embodiments, an enzyme is printed on the surface
of the solid support and a plurality of different substances is
printed on top of the enzyme. In certain embodiments, a plurality
of different enzymes is printed on the surface of the solid support
and the plurality of different substances is printed on top of the
enzymes. In a specific embodiment, all possible protein-substance
combinations are present on a single microarray. In certain, more
specific, embodiments, the substances and/or the enzymes are
purified.
[0067] In certain embodiments, the plurality of proteins consists
of different proteins that are derived from the same source or the
same species, e.g., human, yeast, mouse, rat, bacteria, and C.
elegans. In certain embodiments, the plurality of proteins consists
of different proteins that are known to have a specific enzymatic
activity. In a specific embodiment, the plurality of enzymes
consists of enzymes such as, but not limited to, Oxidoreductases,
Transferases, Hydrolases, Lyases, Isomerases, and Ligases. In more
specific embodiments, an enzyme can be a kinase, a protease, a
phosphatase, a hydrolase, a RNAse, a DNAse, a tryptase, a
phospholipase, or a glycosydase. In certain other embodiments, the
plurality of protein on the microarray consists of different
proteins, wherein the proteins can be derived from different
sources or from different species and where the proteins may have
different or unknown enzymatic activity. In certain embodiments,
proteins with homologies to an enzyme of interest are used with the
invention.
[0068] A substance and a protein can be immobilized on the surface
of the solid support by any method known to the skilled artisan. In
certain embodiments, a substance and/or a protein are directly
immobilized on a glass surface. In certain embodiments, the surface
of the solid support is treated with an aldehyde before a substance
and/or protein is immobilized on the surface. Methods for
immobilizing substrate and protein on the solid support are
described in more detail in section 5.1.
[0069] Any kind of enzymatic reaction known to the skilled artisan
can be used with the methods of the invention. Any group of enzymes
that catalyzes a specific biochemical reaction can be used with the
invention. Any substance can be used with the methods of the
invention. A substance can be a candidate substrate or a known
substrate of an enzymatic reaction of interest. Any substrate can
be used with the methods of the invention. A substrate of an
enzymatic reaction can be, but is not limited to, a proteinaceous
substance (e.g., a protein or a peptide), an organic small
molecule, an inorganic molecule, a nucleic acid (e.g., RNA or DNA),
a lipid, or a carbohydrate.
[0070] In certain embodiments, if enzymes that catalyze a specific
reaction are to be identified, a plurality of different proteins is
printed on the surface of the solid support together with a
substrate that is known to be used in the specific reaction,
wherein each protein is immobilized at a different position of the
microarray. In other embodiments, if a substrate that is used by a
specific enzyme is to be identified, a plurality of different
substances (i.e., candidate substrates) is printed on the surface
of the solid support together with a specific enzyme that is known
to catalyze the specific reaction, wherein each substance is
immobilized at a different position of the microarray. Any method
known to the skilled artisan can be used to visualize and to
quantify the enzymatic reaction. For a more detailed description of
enzymatic reactions and their visualization see section 5.2.
[0071] In certain embodiments, a substance and a protein are
immobilized on the surface of a solid support within a well. In
certain embodiments, each well on the solid support contains at
least one protein and at least one substance such that protein and
substance are in proximity sufficient for the occurrence of an
enzymatic reaction between the substance and the protein. In other
embodiments, a plurality of different proteins or different
substances is printed onto the surface of the solid support such
that each well harbors a plurality of different proteins or
substances. In certain, more specific embodiments, the plurality of
proteins or substances is organized in a positionally addressable
array on the surface within a well. The solid support, e.g., a
slide, can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,
50, 100, 1,000 or at least 10,000 wells. The performance of the
enzymatic reaction on a solid support with wells has the advantage
that different reaction solutions can be added at the same time
onto one solid support (e.g., on one slide).
[0072] In certain specific embodiments, the bottom surface of a
well is coated with a substrate of an enzymatic reaction, wherein
the substrate is immobilized on the surface, and a plurality of
different proteins, e.g., enzymes, are immobilized on the bottom
surface of the well. The substrate and the proteins are in
proximity with each other sufficient for the occurrence of an
enzymatic reaction. In more specific embodiments, each protein of
the plurality of proteins is immobilized at a different position of
the bottom surface of the well in a positionally addressable
fashion.
[0073] In certain embodiments, the proteins of the plurality of
proteins are derived from a single species. In other embodiments,
the proteins of the plurality of proteins are derived from
different species. In more specific embodiments, the proteins of
the plurality of proteins are derived from a prokaryotic organism.
In other embodiments, the proteins of the plurality of proteins are
derived from an organism such as, but not limited to, yeast,
Caenorhabditis elegans, Drosophila melanogaster, mouse, rat, horse,
chimpanzee, or human.
[0074] In a specific embodiment, the enzyme assay of the invention
can be used to analyze the activity of enzymes in a particular
biological sample. This method is useful for, e.g., defining a
pathological state of a cell based on the level of enzyme activity
as opposed to abundance of mRNA or protein. In specific
embodiments, enzymes whose activity is upregulated or downregulated
in a preneoplastic, a neoplastic or a cancerous cell can be
identified. Enzymes whose activity is modulated in a cell of a
specific disease or disorder compared to a normal cell are
candidates for drug targets to identify drugs for treating the
disease or disorder.
[0075] In certain embodiments, a plurality of different substances
is immobilized on the surface of a solid support and the extract of
a cell is also immobilized on the surface of the solid support such
that at least one substance of the plurality of different
substances is in proximity with the extract sufficient for the
occurrence of an enzymatic reaction between the substance and the
protein. In a specific embodiment, at least one substance of the
plurality of different substances is a known substrate of an
enzymatic reaction. In certain embodiments, the different
substances are organized in a positionally addressable array. This
embodiment is useful for assessing enzymatic activities in a
particular type of cell, wherein type of cell can refer to
developmental state of the cell, stage of the cell cycle in the
cell, or whether the cell is derived from a pathological tissue,
e.g., is neoplastic or cancerous. In this embodiment, enzymatic
activity is defined by the substrate used and the enzymatic
reaction performed. In certain, more specific embodiments, the
plurality of different substances is immobilized several times at
different positions of the surface of the solid support. In certain
embodiments, extracts from different types of cells are immobilized
at the different positions such that each plurality or at least
some of the pluralities of different substances are in contact with
a different cellular extract. In certain embodiments, each
plurality or at least some of the pluralities of different
substances are in proximity with cellular extract from the same
type of cell sufficient for the occurrence of an enzymatic reaction
between the substances of the pluralities and the proteins of the
cellular extract. In certain embodiments, different reaction
mixtures, i.e., reaction mixtures providing different conditions
and/or cofactors, are contacted with the different pluralities of
different substances.
[0076] The invention also relates to protein microarrays. In
certain embodiments the invention provides a positionally
addressable array comprising at least one known enzyme and at least
one candidate substrate of the enzyme, wherein (i) the enzyme and
the substrate are immobilized on the surface of a solid support;
(ii) the enzyme and the substrate are in proximity sufficient for
the occurrence of the enzymatic reaction catalyzed by the enzyme
between the enzyme and the substrate; and (iii) the enzyme and the
substrate are not identical to each other. In other embodiments,
the positionally addressable array of the invention comprises at
least one known substrate of an enzymatic reaction and at least one
candidate enzyme for the catalysis of the enzymatic reaction,
wherein (i) the enzyme and the substrate are immobilized on the
surface of a solid support; (ii) the enzyme and the substrate are
in proximity sufficient for the occurrence of the enzymatic
reaction between the enzyme and the substrate; and (iii) the enzyme
and the substrate are not identical to each other. In even other
embodiments, a positionally addressable array comprises at least
one known substrate of an enzymatic reaction and at least one
enzyme that is known to catalyze the enzymatic reaction, wherein
(i) the enzyme and the substrate are immobilized on the surface of
a solid support; (ii) the enzyme and the substrate are in proximity
sufficient for the occurrence of the enzymatic reaction between the
enzyme and the substrate; and (iii) the enzyme and the substrate
are not identical to each other.
[0077] In certain embodiments, a plurality of proteins and a
substance are immobilized on the microarrays of the invention. The
plurality of proteins can be a selection of proteins, such as, but
not limited to, proteins derived from a single species, proteins of
a particular enzymatic activity, proteins with regions of homology
to an enzyme of interest, and proteins derived from a specific
cellular extract. The microarray of the invention can be coated
with a substance, or the substance can be printed on different
spots of the surface of the solid support and the proteins of the
plurality of proteins are printed on top of the substance. In
certain more specific embodiments, the substance is a known
substrate of the enzymatic reaction to be assayed. In certain, more
specific embodiments, each protein of the plurality of proteins is
immobilized at a different position of the surface of the solid
support. Alternatively, the plurality of proteins is printed first
and the substance is printed subsequently on top of the proteins.
In certain embodiments, the plurality of proteins is organized in a
positionally addressable array.
[0078] In other embodiments, a plurality of substances and an
enzyme are immobilized on the microarrays of the invention. The
plurality of substances can be a selection of proteins, peptides,
sugars, polysaccharides, small organic molecules, inorganic
molecules, DNA or RNA. The microarray of the invention can be
coated with the enzyme, or the enzyme can be printed on different
spots of the surface of the solid support and the substances of the
plurality of substances are printed on top of the enzyme.
Alternatively, the plurality of substances is printed first and the
enzyme is printed subsequently on top of the substances.
[0079] In certain embodiments, the microarrays of the invention
have wells. In certain embodiments, at least one well is pre-coated
or pre-printed with a substance and a plurality of different
proteins is printed on the surface of the solid support in the well
such that a substance and a protein are in proximity with each
other sufficient for the occurrence of an enzymatic reaction
between the protein and the substance. In certain embodiments, at
least one well is pre-coated or pre-printed with an enzyme and a
plurality of different substances is printed on the surface of the
solid support in the well such that a substance and an enzyme are
in proximity with each other sufficient for the occurrence of an
enzymatic reaction between the protein and the substance. In
certain, more specific embodiments, the substances are potential
substrates of the enzyme. In other embodiments, the substances are
known substrates of the enzyme.
[0080] In certain embodiments, each well of a microarray of the
invention has the same combination of substances and proteins
immobilized to the surface of the solid support within the well. In
this embodiment, each well of the microarray can be filled with a
different reaction buffer such that the enzymatic reaction(s) can
be monitored under a plurality of different reaction conditions; in
the presence and absence, respectively, of a plurality of different
test molecules; or in the presence and absence, respectively, of
different cofactors.
[0081] The invention also provides kits for carrying out the assay
regimens of the invention and for manufacturing the microarrays of
the invention. In a specific embodiment, kits of the invention
comprise one or more arrays of the invention. Such kits may further
comprise, in one or more containers, reagents useful for assaying
biological activity of a protein or molecule, reagents useful for
assaying interaction of a substrate and a protein or enzyme,
reagents useful for assaying the biological activity of a protein
or molecule having a biological activity of interest. The reagents
useful for assaying biological activity of a protein or molecule,
or assaying interactions between a probe and a protein or molecule,
can be contained in each well or selected wells on the protein
chip. Such reagents can be in solution or in solid form. The
reagents may include either or both the proteins or molecules and
the substrates required to perform the assay of interest.
[0082] In one embodiment, a kit comprises one or more protein
microarrays of the invention. In certain embodiments, the proteins
and substrates are already immobilized onto the surface of the
solid support. In another embodiment, reagents are provided in the
kit that can be used for immobilizing substrate and protein onto
the surface of the solid support.
[0083] In certain embodiments, the substrate is different from the
proteins of the plurality of proteins. In certain embodiments, the
substrate is different from the enzyme.
[0084] In certain embodiments, the invention provides a method for
assaying an enzymatic reaction, the method comprising: (a)
incubating at least one protein, at least one first substance, and
at least one second substance under conditions conducive to the
occurrence of an enzymatic reaction between the protein and the
first or the second substance, wherein (i) the protein, the first
substance and the second substance are immobilized on the surface
of a solid support; (ii) the protein, the first substance and the
second substance are in proximity sufficient for the occurrence of
said enzymatic reaction; (iii) the protein and the first substance
are not identical and (iv) the protein and the second substance are
not identical; and (b) determining whether said enzymatic reaction
occurs.
[0085] 5.1. Solid Support and Immobilization Of Substrate and
Protein
[0086] In the methods and microarrays of the invention, at least
one substance and at least one protein are immobilized on the
surface of a solid support such that substance and protein are in
proximity sufficient for the occurrence of an enzymatic reaction.
The substance is a candidate substrate or a known substrate of the
enzymatic reaction. The protein is a candidate enzyme or an enzyme
known to catalyze the enzymatic reaction of interest.
[0087] The substance and the protein can be immobilized to the
surface of the solid support by any method known to the skilled
artisan. In certain embodiments, the substance is immobilized
before the protein is immobilized. In other embodiments, the
protein is immobilized before the substance is immobilized. The
suitability of a specific method of immobilizing a protein or a
substrate may depend on the molecular nature of the protein or
substance. If the substrate is a proteinaceous substance, e.g., a
protein or a peptide, any method known to the skilled artisan can
be used to immobilize a protein to the surface of a solid support.
If the substance is not a proteinaceous substance, any method known
to the skilled artisan can be used to immobilize a molecule of that
type of molecules to surface of a solid support.
[0088] In certain embodiments of the invention, the substance and
the protein are immobilized on the surface of the solid support
such that substance and protein are in proximity with each other
sufficient for the occurrence of the enzymatic reaction to be
assayed. In certain embodiments of the invention, the substance and
the protein are immobilized on the surface of the solid support
such that substance and protein are in physical contact with each
other.
[0089] In certain embodiments, the substance is purified. In
certain embodiments, the protein is purified. In certain
embodiments, the substance and the protein are purified.
[0090] In certain embodiments, the surface of a solid support is
coated or printed with a mixture of at least 2, 3, 4, 5, 10, 15,
20, 25, 50 or 100 different substances. In certain embodiments, the
surface of a solid support is coated or printed with a mixture of
at most 2, 3, 4, 5, 10, 15, 20, 25, 50 or 100 different substances.
In certain embodiments, a plurality of different mixtures of
substances is immobilized on the surface of the solid support.
[0091] The solid support can be constructed from materials such as,
but not limited to, silicon, glass, quartz, polyimide, acrylic,
polymethylmethacrylate (LUCITE.RTM.), ceramic, gold,
nitrocellulose, amorphous silicon carbide, polystyrene, and/or any
other material suitable for microfabrication, microlithography, or
casting. For example, the solid support can be a hydrophilic
microtiter plate (e.g., MILLIPORE.TM.) or a nitrocellulose-coated
glass slide. In a specific embodiment, the solid support is a
nitrocellulose-coated glass slide. Nitrocellulose-coated glass
slides for making protein (and DNA) microarrays are commercially
available (e.g., from Schleicher & Schuell (Keene, N.H.), which
sells glass slides coated with a nitrocellulose based polymer (Cat.
no. 10 484 182)). In a specific embodiment, each protein is spotted
onto the nitrocellulose-coated glass slide using an OMNIGRID.TM.
(GeneMachines, San Carlos, Calif.). The present invention
contemplates other solid supports useful for constructing a protein
chip, some of which are disclosed, for example, in International
Patent Application publication WO 01/83827 which is incorporated
herein by reference in its entirety.
[0092] In one embodiment, the solid support is a flat surface such
as, but not limited to, a glass slide. Dense protein arrays can be
produced on, for example, glass slides, such that assays for the
presence, amount, and/or functionality of proteins can be conducted
in a high-throughput manner.
[0093] In certain, more specific embodiments, the solid support is
a glass slide that has been pre-treated with an aldhyde, such as
paraformaldehyde or formaldehyde. In certain embodiments, the solid
support is an aldehyde treated slide is obtained from TeleChem
International, Inc. In other embodiments, the solid support is a
nitrocellulose coated slide (Schleicher & Schuell). In other
embodiments, the solid support is coated with an amino-silane
surface (GAPS slide obtained from Corning.RTM.).
[0094] In certain embodiments, after immobilizing the substances
and the proteins, the chip is blocked. Any blocking agent known to
the skilled artisan can be used with the methods of the invention.
In a specific embodiment, Bovine Serum Albumin, glycine or a
detergent (e.g., Tween20) can be used as a blocking agent. In
certain other embodiments, the chips are not blocked.
[0095] In a particular embodiment, the solid support comprises a
silicone elastomeric material such as, but not limited to,
polydimethylsiloxane ("PDMS"). An advantage of silicone elastomeric
materials is their flexible nature.
[0096] In another particular embodiment, the solid support is a
silicon wafer. The silicon wafer can be patterned and etched (see,
e.g., G. Kovacs, 1998, Micromachined Transducers Sourcebook,
Academic Press; M. Madou, 1997, Fundamentals of Microfabrication,
CRC Press). The etched wafer can also be used to cast the
microarrays to be used with the invention.
[0097] Accordingly, in certain embodiments, the plurality of
proteins is applied to the surface of a solid support, wherein the
density of the sites at which protein are applied is at least 1
site/cm.sup.2, 2 sites/cm.sup.2, 5 sites/cm.sup.2, 10
sites/cm.sup.2, 25 sites/cm.sup.2, 50 sites/cm.sup.2, 100
sites/cm.sup.2, 1000 sites/cm.sup.2, 10,000 sites/cm.sup.2, 100,000
sites/cm.sup.2, 1,000,000 sites/cm.sup.2, 10,000,000
sites/cm.sup.2, 25,000,000 sites/cm.sup.2, 10,000,000,000
sites/cm.sup.2, or 10,000,000,000,000 sites/cm.sup.2. Each
individual protein sample is preferably applied to a separate site
on the chip. In certain specific embodiments, the identities of the
protein(s) at each site on the chip is/are known. In certain other
embodiments, a plurality of substances is applied to the surface of
a solid support, wherein the density of the sites at which
substances are applied is at least 1 site/cm.sup.2, 2
sites/cm.sup.2, 5 sites/cm.sup.2, 10 sites/cm.sup.2, 25
sites/cm.sup.2, 50 sites/cm.sup.2, 100 sites/cm.sup.2, 1000
sites/cm.sup.2, 10,000 sites/cm.sup.2, 100,000 sites/cm.sup.2,
1,000,000 sites/cm.sup.2, 10,000,000 sites/cm.sup.2, 25,000,000
sites/cm.sup.2, 10,000,000,000 sites/cm.sup.2, or
10,000,000,000,000 sites/cm.sup.2. Each individual protein sample
is preferably applied to a separate site on the chip. In certain
specific embodiments, the identities of the proteins at each site
on the chip are known, i.e., the chip is a positionally addressable
array.
[0098] In certain embodiments, a plurality of different proteins is
applied to the surface, wherein the surface is either pre-coated
with a substance or pre-printed with substance. If the surface is
pre-printed with a substance, care should be taken that each of the
different proteins is printed on top of the sites where a substance
is present. In certain other embodiments, a plurality of different
substances is applied to the surface, wherein the surface is either
pre-coated with an enzyme or pre-printed with an enzyme. If the
surface is pre-printed with an enzyme, care should be taken that
each of the different substances is printed on top of the sites
where enzyme is present. The substrate can be a candidate substrate
for the enzymatic reaction to be assayed.
[0099] In certain embodiments, a substance and an enzyme are
immobilized on the surface of a solid support, wherein the solid
support has wells. In certain embodiments, a plurality of different
enzymes or different substances is printed on the surface of the
solid support such that each feature of the microarray is in a
different well. In other embodiments, a plurality of different
enzymes or different substances is printed onto the surface of the
solid support such that each well harbors a plurality of different
proteins or substrates. The performance of the enzymatic reaction
on a solid support with wells has the advantage that different
reaction solutions can be added at the same time onto one solid
support (e.g., on one slide). Another advantage of wells over flat
surfaces is an increased signal-to-noise ratio. Wells allow the use
of larger volumes of reaction solution in a denser configuration,
and therefore greater signal is possible. Furthermore, wells
decrease the rate of evaporation of the reaction solution from the
chip as compared to flat surface arrays, thus allowing longer
reaction times. Another advantage of wells over flat surfaces is
that the use of wells permit association studies using a specific
volume of reaction volume for each well on the chip, whereas the
use of flat surfaces usually involves indiscriminate probe
application across the whole surface. The application of a defined
volume of reaction buffer can be important if a reactant that is
supplied in the reaction buffer is being depleted during the course
of the reaction. In such a scenario, the application of a defined
volume allows for more reproducible results. The use of
microlithographic and micromachining fabrication techniques (see,
e.g., International Patent Application publication WO 01/83827,
which is incorporated herein by reference in its entirety) can be
used to create well arrays with a wide variety of dimensions
ranging from hundreds of microns down to 100 nm or even smaller,
with well depths of similar dimensions. In one embodiment, a
silicon wafer is micromachined and acts as a master mold to cast
wells of 400 .mu.m diameter that are spaced 200 .mu.m apart, for a
well density of about 277 wells per cm.sup.2, with individual well
volumes of about 30 nl for 100 .mu.m deep wells (see, e.g.,
International Patent Application publication WO 01/83827, which is
incorporated herein by reference in its entirety).
[0100] In certain embodiments, the wells of a microarray of the
invention have depth. In other embodiments, the wells of a
microarray of the invention do not have depth. In a nonlimiting
example, the different wells are separated by barriers wherein the
barrier comprises a different surface material than the surface
material of the well. E.g. the wells are constituted by an area on
the solid support that is a glass surface and the barriers are
constituted by a surface material such as teflon. Such slides can
be obtained, e.g., from Erie Scientific Company, NH. Without being
bound by theory, the difference in surface tension provided by the
different surface materials ensures that a liquid from one well
will not leak into a neighboring well.
[0101] In one embodiment, the solid support comprises gold. In a
preferred embodiment, the solid support comprises a gold-coated
slide. In another embodiment, the solid support comprises nickel.
In another preferred embodiment, the solid support comprises a
nickel-coated slide. Solid supports comprising nickel are
advantageous for purifying and attaching fusion proteins having a
poly-histidine tag ("His tag"). In another embodiment, the solid
support comprises nitrocellulose. In another preferred embodiment,
the solid support comprises a nitrocellulose-coated slide.
[0102] The proteins and substances can be bound directly to the
solid support, or can be attached to the solid support through a
linker molecule or compound. The linker can be any molecule or
compound that derivatizes the surface of the solid support to
facilitate the attachment of proteins and/or substrates to the
surface of the solid support. The linker may covalently or
non-covalently bind the proteins or substrates to the surface of
the solid support. In addition, the linker can be an inorganic or
organic molecule. In certain embodiments, the linker may be a
silane, e.g., sianosilane, thiosilane, aminosilane, etc. Compounds
useful for derivatization of a protein chip are also described in
International Patent Application publication WO 01/83827, which is
incorporated herein by reference in its entirety.
[0103] Accordingly, in one embodiment, the proteins and/or
substrates are bound non-covalently to the solid support (e.g., by
adsorption). Proteins and/or substrates that are non-covalently
bound to the solid support can be attached to the surface of the
solid support by a variety of molecular interactions such as, for
example, hydrogen bonding, van der Waals bonding, electrostatic, or
metal-chelate coordinate bonding. In a particular embodiment,
proteins and/or substrates are bound to a poly-lysine coated
surface of the solid support. In addition, as described above, in
certain embodiments, the proteins and/or substrates are bound to a
silane (e.g., sianosilane, thiosilane, aminosilane, etc.) coated
surface of the solid support.
[0104] In addition, crosslinking compounds commonly known in the
art, e.g. homo- or heterofunctional crosslinking compounds (e.g.,
bis[sulfosuccinimidyl]suberate,
N-[gamma-maleimidobutyryloxy]succinimide ester, or
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide), may be used to
attach proteins and/or substrates to the solid support via covalent
or non-covalent interactions.
[0105] In another embodiment, proteins and/or substrates of the
protein chip are bound covalently to the solid support. In other
embodiments, proteins and/or substrates can be bound to the solid
support by receptor-ligand interactions, which include interactions
between antibodies and antigens, DNA-binding proteins and DNA,
enzyme and substrate, avidin (or streptavidin) and biotin (or
biotinylated molecules), and interactions between lipid-binding
proteins and phospholipids (or membranes, vesicles, or liposomes
comprising phospholipids).
[0106] Purified proteins and/or substrates can be placed on an
array using a variety of methods known in the art. In one
embodiment, the proteins and/or substrates are printed onto the
surface of a solid support. In a further embodiment, the proteins
and/or substrates are attached to the solid support using an
affinity tag. In a specific embodiment, an affinity tag different
from that used to purification of the protein or substrate is used
for immobilizing the protein or substrate. If two different tags
are used further purification is achieved when building the protein
array.
[0107] In a specific embodiment, proteins and/or substrates are
expressed as fusion proteins having at least one heterologous
domain with an affinity for a compound that is attached to the
surface of the solid support. Suitable compounds useful for binding
fusion proteins onto the solid support (i.e., acting as binding
partners) include, but are not limited to, trypsin/anhydrotrypsin,
glutathione, immunoglobulin domains, maltose, nickel, or biotin and
its derivatives, which bind to bovine pancreatic trypsin inhibitor,
glutathione-S-transferase, Protein A or antigen, maltose binding
protein, poly-histidine (e.g., HisX6 tag), and avidin/streptavidin,
respectively. For example, Protein A, Protein G and Protein A/G are
proteins capable of binding to the Fe portion of mammalian
immunoglobulin molecules, especially IgG. These proteins can be
covalently coupled to, for example, a Sepharose.RTM. support to
provide an efficient method of purifying fusion proteins having a
tag comprising an Fc domain. In a specific embodiment, the proteins
are bound to the solid support via His tags, wherein the solid
support comprises a flat surface. In a preferred embodiment, the
proteins are bound to the solid support via His tags, wherein the
solid support comprises a nickel-coated glass slide.
[0108] In certain embodiments, proteins and/or substrates are
expressed as fusion proteins, wherein the protein and/or substrate
is fused to a bifunctional tag. In an example of such an
embodiment, the protein and/or substrate is fused to an intein and
a chitin binding domain. In a more specific embodiment, the
proteins and/or substrates are expressed using the IMPACT.TM.-CN
system from New England Biolabs Inc. In the presence of thiols such
as DTT, b-mercaptoethanol or cysteine, the intein undergoes
specific self-cleavage which releases the target protein from the
chitin-bound intein tag.
[0109] The protein chips to be used with the present invention are
not limited in their physical dimensions and can have any
dimensions that are useful. Preferably, the protein chip has an
array format compatible with automation technologies, thereby
allowing for rapid data analysis. Thus, in one embodiment, the
protein microarray format is compatible with laboratory equipment
and/or analytical software. In a preferred embodiment, the protein
chip is the size of a standard microscope slide. In another
preferred embodiment, the protein chip is designed to fit into a
sample chamber of a mass spectrometer.
[0110] In specific embodiments, protein and/or substrate are
applied to a flat surface, such as, but not limited to, glass
slides. Proteins and/or substrate are bound covalently or
non-covalently to the flat surface of the solid support. The
proteins and/or substrate can be bound directly to the flat surface
of the solid support, or can be attached to the solid support
through a linker molecule or compound. The linker can be any
molecule or compound that derivatizes the surface of the solid
support to facilitate the attachment of proteins and/or substrate
to the surface of the solid support. The linker may covalently or
non-covalently bind the proteins and/or substrate to the surface of
the solid support. In addition, the linker can be an inorganic or
organic molecule. Specific linkers are compounds with free amines.
Preferred among linkers is 3-glycidooxypropyltrimethoxysilane
(GPTS).
[0111] In a specific embodiment, by way of example and not
limitation, proteins are immobilized on the solid support using the
following procedure: Briefly, after washing with 100% ethanol
(EtOH) three times at room temperature, the chips (e.g., chips made
of polydimethylsiloxane or glass slides) are immersed in 1% GPTS
solution (95% ethanol (EtOH), 16 mM acetic acid (HOAc)) with
shaking for 1 hr at room temperature. After three washes with 95%
EtOH, the chips are cured at 135.degree. C. for 2 hrs under vacuum.
Cured chips can be stored in dry Argon for months 12. To attach
proteins and substrates to the chips, protein solutions are added
to the wells and incubated on ice for 1 to 2 hours. After rinsing
with cold HEPES buffer (10 mM HEPES, 100 mM NaCl, pH 7.0) three
times, the wells are blocked with 1% BSA in PBS (Sigma, USA) on ice
for >1 hr. Because of the use of GPTS, any reagent containing
primary amine groups is avoided.
[0112] In another embodiment, protein-containing cellular material,
such as but not limited to vesicles, endosomes, subcellular
organelles, and membrane fragments, can be placed on the protein
chip. In another embodiment, a whole cell is placed on the protein
chip. In a further embodiment, the protein, protein-containing
cellular material, or whole cell is attached to the solid support
of the protein chip. In a specific embodiments, the protein,
protein-containing cellular material, or whole cell is attached to
the surface of the solid support that is coated or preprinted with
substrate.
[0113] Furthermore, proteins, substrate, protein- or
substrate-containing cellular material, or cells can be embedded in
artificial or natural membranes prior to or at the time of
placement on the protein chip. Embedding enzymes in membranes is
the preferred embodiment, if the enzyme assumes its enzymatically
active conformation preferentially in a membrane. In another
embodiment, proteins, protein-containing cellular material, or
cells can be embedded in extracellular matrix component(s) (e.g.,
collagen or basal lamina) prior to or at the time of placement on
the protein chip.
[0114] The proteins or substrates are bound covalently or
non-covalently to the surface of the solid support in the wells. In
more specific embodiments, the protein is bound covalently to the
surface and the substrate is bound non-covalently to the surface.
In other embodiments, the protein is bound non-covalently to the
surface and the substrate is bound covalently to the surface. In
other embodiments, both substrate and protein are bound covalently
to the surface. In other embodiments, both substrate and protein
are bound non-covalently to the surface. The proteins or substrates
can be bound directly to the surface of the solid support, or can
be attached to the solid support through a linker molecule or
compound. The linker can be any molecule or compound that
derivatizes the surface of the solid support to facilitate the
attachment of proteins or substrates to the surface of the solid
support. The linker may covalently bind the proteins or substrates
to the surface of the solid support or the linker may bind via
non-covalent interactions. In addition, the linker can be an
inorganic or organic molecule. Preferred linkers are compounds with
free amines. Most preferred among linkers is
3-glycidooxypropyltrimethoxysilane (GPTS).
[0115] Proteins or substrates which are non-covalently bound to the
surface of the solid support may utilize a variety of molecular
interactions to accomplish attachment to surface of the solid
support such as, for example, hydrogen bonding, van der Waals
bonding, electrostatic, or metal-chelate coordinate bonding.
Further, DNA-DNA, DNA-RNA and receptor-ligand interactions are
types of interactions that utilize non-covalent binding. Examples
of receptor-ligand interactions include interactions between
antibodies and antigens, DNA-binding proteins and DNA, enzyme and
substrate, avidin (or streptavidin) and biotin (or biotinylated
molecules), and interactions between lipid-binding proteins and
phospholipid membranes or vesicles. For example, proteins and/or
substrates can be expressed with fusion protein domains that have
affinities for a binding partner that is attached to the surface of
the solid support. Suitable binding partners for fusion protein
binding include trypsin/anhydrotrypsin, glutathione, immunoglobulin
domains, maltose, nickel, or biotin and its derivatives, which bind
to bovine pancreatic trypsin inhibitor, glutathione-S-transferase,
antigen, maltose binding protein, poly-histidine (e.g., HisX6 tag),
and avidin/streptavidin, respectively.
[0116] In certain embodiments, the proteins and/or the substrate is
immobilized to the solid support via a peptide tag, wherein the
affinity binding partner for the tag is attached (covalently or
non-covalently) to the solid support. For a more detailed
description of peptide tags see section 5.5.1.
[0117] In certain embodiments, a protein is immobilized directly on
the surface of the solid support. In other embodiments, a protein
is immobilized via a linker molecule to the solid support. In
certain, more specific embodiments, the distance between a protein
and the surface of a solid support is at most 0.1 nm, 1 nm, 5 nm,
10 nm, 15 nm, 25 nm, 50 nm, 100 nm, 1 .mu.m or at most 5 .mu.m. In
certain embodiments, the distance between the protein and the
surface of the solid support is at least 0.1 nm, 1 nm, 5 nm, 10 nm,
15 nm, 25 nm, 50 nm, 100 nm, 1 .mu.m or at least 5 .mu.m. In
certain embodiments, a protein is immobilized to the underivatized
surface of a solid support. In a more specific embodiment, a
protein is immobilized to the underivitized glass surface of a
solid support.
[0118] In certain embodiments, the substance is immobilized
directly on a surface of a solid support. In other embodiments, a
substance is immobilized via a linker molecule to a solid support.
In certain, more specific embodiments, the distance between a
substance and the surface of a solid support is at most 0.1 nm, 1
nm, 5 nm, 10 nm, 15 nm, 25 nm, 50 nm, 100 nm, 1 .mu.m or at most 5
.mu.m. In certain embodiments, the distance between a substance and
the surface of a solid support is at least 0.1 nm, 1 nm, 5 nm, 10
nm, 15 nm, 25 nm, 50 nm, 100 nm, 1 .mu.m or at least 5 .mu.m. In
certain embodiments, a substance is immobilized to the
underivatized surface of a solid support. In a more specific
embodiment, the substance is immobilized to the underivitized glass
surface of a solid support.
[0119] In certain embodiments, a substance and a protein are
immobilized directly on the surface of the solid support. In other
embodiments, a substance and a protein are immobilized via a linker
molecule to the solid support. In certain, more specific
embodiments, the distance between a substance and the surface of
the solid support and the distance between a protein and the
surface of the solid support (i.e., the length of the linker
molecule, or the distance by which the linker distances the
substance or the protein from the solid support) is at most 0.1 nm,
1 nm, 5 nm, 10 nm, 15 nm, 25 nm, 50 nm, 100 nm, 1 .mu.m or at most
5 .mu.m. In certain embodiments, the distance between a substance
and the surface of the solid support and the distance between a
protein and the surface of the solid support is at least 0.1 nm, 1
nm, 5 nm, 10 nm, 15 nm, 25 nm, 50 nm, 100 nm, 1 .mu.m or at least 5
.mu.m. In certain embodiments, a substance and a protein are
immobilized to the underivatized surface of the solid support. In a
more specific embodiment, a substance and a protein are immobilized
to the underivitized glass surface of a solid support.
[0120] The solid support can have a porous or a non-porous
surface.
[0121] An aspect to be considered when choosing the surface
chemistry for immobilizing substance and a protein are background
signals created by the surface (see, e.g., FIG. 2 of section
6.1).
[0122] Proteins can be immobilized in many ways on a surface. In
certain embodiments, a substrate or a protein can be immobilized
reversibly. In other embodiments, a substrate or a protein can be
immobilized irreversibly. The goal of immobilizing a substrate and
a protein is to retain the protein and the substrate in a defined
region on the microarray. The protein and/or the substrate can be
encapsulated or entrapped in a porous surface or a vesicle. The
protein and/or the substrate can be kinetically trapped but has
free molecules in equilibrium with surface-bound ones.
[0123] In certain embodiments, the different proteins and/or the
different substances on the surface of a solid support are present
in approximately equimolar amounts. Without being bound by theory,
using approximately equimolar amounts facilitates the
quantification of the results obtained.
[0124] In certain embodiments of the invention, the amount of a
protein or a substance is present on the surface of a solid support
is at least 10.sup.-12 mol, 10.sup.-11 mol, 10.sup.-10 mol,
10.sup.-9 mol, 10.sup.-8 mol, 10.sup.-7 mol, 10.sup.-6 mol,
10.sup.-5 mol, 10.sup.-4 mol, 10.sup.-3 mol, 10.sup.-2 mol, or at
least 10.sup.-1 mol. In certain embodiments of the invention, the
amount of a protein or a substance is present on the surface of a
solid support is at most 10.sup.-12 mol, 10.sup.-11 mol, 10.sup.-10
mol, 10.sup.-9 mol, 10.sup.-8 mol, 10.sup.-7 mol, 10.sup.-6 mol,
10.sup.-5 mol, 10.sup.-4 mol, 10.sup.-3 mol, 10.sup.-2 mol, or at
least 10.sup.-1 mol.
[0125] Illustrative examples of immobilizing a protein and a
substrate include, but are not limited to,
[0126] 1. Immobilization by specific covalent bonds, such as
disulfide with a cysteine, or non-specific covalent bonds, such as
a Schiff base, formed between a protein or a substance and the
surface of the solid support (e.g., a slide).
[0127] 2. Immobilization by adsorption of a protein or a substance
directly onto the surface of the solid support.
[0128] 3. Immobilization by specific non-covalent interactions
between a substance or a protein and the surface, such as
His-tagged proteins or substances and Nickel surfaces.
[0129] 4. Immobilization indirectly by interactions of a protein or
a substance with immobilized molecules, including proteins, lipids,
nucleic acids and carbohydrates.
[0130] 5. The interactions of a protein or a substance with
immobilized molecules can be specific, such as antibody/antigen or
streptavidin/biotin.
[0131] 6. The interactions of a protein or a substance with
immobilized molecules can be non-specific.
[0132] 7. Immobilization by cross linking to a matrix on the
slide.
[0133] 8. Immobilization by entrapment in a matrix on the
slide.
[0134] 9. The matrix can be made of polymers. The polymerization
and/or the cross linking can occur before, during and after the
printing of proteins.
[0135] 10. The matrix can be made of interactions of non-covalent
natures, such as hydrogen bonds and van der Waals interactions,
between the same or different types of molecules.
[0136] 11. A protein or a substance to be immobilized can be part
of the matrix formation.
[0137] 12. Immobilization by encapsulation of a protein or a
substance in molecular-scale compartments, such as liposomes,
vesicles or micelles, which are covalently or non-covalently
attached to a surface.
[0138] 13. Immobilization by protein aggregation, cross-linking,
precipitation or denaturation on the surface of a solid
support.
[0139] In certain embodiments, substrate and protein are
immobilized by different procedures. In certain other embodiments,
substrate and protein are immobilized by the same procedure.
[0140] Covalent bonding or other strong interactions between a
protein and the surface of a solid support may modify the structure
and thus function of a protein. Thus, the skilled artisan can,
e.g., by means of structural prediction programs, available
structures of proteins or experimental determination of a structure
determine which region of a protein is best suited to be in contact
with the surface or the linker. In an illustrative embodiment, a
protein is known to have two structural domains, a first domain
with catalytic activity and a second domain. In a specific
embodiment, the second domain is linked to the surface of the solid
support. In another embodiment, the first domain is linked to the
surface of the solid support. Without being bound by theory,
immobilization directly through the domain with the catalytic
activity may inhibit activity. Immobilization of catalytic domains
may not be desirable. Instead, immobilization through a fused
domain or protein may offer better activity.
[0141] Other factors to be considered in generating the microarrays
to be used with the methods of the invention are: Enzymatic
activities increase with the amounts of enzymes and substrates.
Higher activities will also result if the effective concentrations
of enzyme and substrate are higher. Proteins may denature at
liquid/solid or air/liquid interface, resulting in less activity.
Restricting enzyme or substrate conformations on a surface may
reduce productive interactions between the molecules. The diffusion
rate of large molecules is low, and the rate of reaction can be
diffusion-limited.
[0142] In certain embodiments, slides with high protein binding
capacities are used to increase local enzyme and/or substance
concentrations. Without being limited by theory, bringing enzymes
and substances into closer proximity may increases the effective
concentrations. Immobilization of a protein or a substance by
non-specific adsorption may denature a protein. Interactions
between slide surface and a protein or a substance may reduce their
diffusion rates. The interactions increase with larger surface
areas as on surfaces made of porous materials or matrices. Further,
entrapment or immobilization using indirect methods may be less
disruptive to the enzymes.
[0143] For the microarray assay to work effectively, the background
signals from labeled molecules need to be minimized. In certain
embodiments, the interactions between the surface and a labeled
molecule that is used in the enzymatic reaction can be blocked with
a non-labeled molecule before or during the enzymatic reaction to
minimize background. The binding kinetics of molecules often depend
on the concentrations of the probe, available slide surface areas
for binding, temperature as well as the specific chemistry. Slides
made of matrices or porous materials have much higher surface areas
and thus potentially more interactions with the labeled
molecules.
[0144] In certain embodiments, surfaces having slower binding
kinetics compared to the assay time may offer better signal to
background.
[0145] In certain embodiments, surfaces with lower protein binding
capacities may reduce background. However, the binding capacity
must be weighed with the sensitivity of the enzymatic assay as a
reduction in enzyme will also reduce signal intensity.
[0146] Other considerations include that surface chemistry also
affects the making of protein microarrays. The surface properties,
such as hydrophobicity, flatness, and homogeneity, influence the
amount of proteins delivered to the slide and the size and
morphology of the spots. These factors will ultimately affect the
assay sensitivity and reproducibility.
[0147] 5.2. Enzymatic Reactions and their Quantification
[0148] In certain embodiments, an enzymatic reaction of interest is
performed wherein a substance and a protein are immobilized on the
surface of a solid support such that the substance and the protein
are in proximity sufficient for the occurrence of the enzymatic
reaction. The reaction is performed by incubating the substance and
the protein in a reaction mixture or reaction buffer that provides
conditions conducive to the occurrence of the enzymatic reaction.
The reaction conditions provided by the reaction buffer or mixture
depend on the type of enzymatic reaction being performed and
include, but are not limited to, salt concentration, detergent
concentration, cofactors and pH. Other reaction conditions, such as
temperature, also depend on the type of enzymatic reaction being
performed.
[0149] Any enzymatic reaction known to the skilled artisan can be
performed with the methods of the invention. If the reaction
involves more than one substrate, at least one substrate is
immobilized, the other substrates can also be immobilized or can be
in solution. In certain embodiments, if the enzymatic reaction
involves one or more co-factors, such as, but not limited to, NAD,
NADH or ATP, such a co-factor can be in solution or can also be
immobilized on the surface of the solid support. Any method known
to the skilled artisan can be used to visualize and quantitate the
activity of the enzyme.
[0150] In certain embodiments, the enzymatic reaction is performed
such that the generation of the product of the reaction results in
the emergence of a detectable signal. In certain embodiments, the
enzymatic reaction is performed such that an increase in
concentration of the product of the reaction results in an increase
of a detectable signal. In other embodiments, the enzymatic
reaction is performed such that an increase in concentration of the
product of the reaction results in a decrease of a detectable
signal. In certain embodiments, the enzymatic reaction is performed
such that an decrease of substrate concentration results in the
increase or decrease of a detectable signal.
[0151] In certain embodiments, standard enzymatic assays that
produce chemiluminescence or fluorescence are performed using a
microarray, wherein enzyme and substrate are immobilized on the
surface of a solid support. Detection and quantification of an
enzymatic reaction can be accomplished using, for example,
photoluminescence, radioactivity, fluorescence using non-protein
substrates, enzymatic color development, mass spectroscopic
signature markers, and amplification (e.g., by PCR) of
oligonucleotide tags. In a specific embodiment, peptides or other
compounds released into solution by the enzymatic reaction of the
array elements can be identified by mass spectrometry.
[0152] The types of assays to detect and quantify the products (or
the decrease of substrate) of an enzymatic reaction fall into
several general categories. Such categories of assays include, but
not limited to: 1) using radioactively labeled reactants followed
by autoradiography and/or phosphoimager analysis; 2) binding of
hapten, which is then detected by a fluorescently labeled or
enzymatically labeled antibody or high affinity hapten ligand such
as biotin or streptavidin; 3) mass spectrometry; 4) atomic force
microscopy; 5) fluorescent polarization methods; 6) rolling circle
amplification-detection methods (Schweitzer et al., 2000,
"Immunoassays With Rolling Circle DNA Amplification: A Versatile
Platform For Ultrasensitive Antigen Detection", Proc. Natl. Acad.
Sci. USA 97:10113-10119); 7) competitive PCR (Fini et al., 1999,
"Development of a chemiluminescence competitive PCR for the
detection and quantification of parvovirus B19 DNA using a
microplate luminometer", Clin Chem. 45(9):1391-6; Kruse et al.,
1999, "Detection and quantitative measurement of transforming
growth factor-beta1 (TGF-beta1) gene expression using a semi-nested
competitive PCR assay", Cytokine 11(2):179-85; Guenthner and Hart,
1998, "Quantitative, competitive PCR assay for HIV-1 using a
microplate-based detection system", Biotechniques 24(5):810-6); 8)
colorimetric procedures; and 9) FRET.
[0153] Useful information also can be obtained, for example, by
performing the assays of the invention with cell extracts. In a
specific embodiment, different substrates of an enzymatic reaction
are immobilized on the surface of a solid support and the proteins
of the cell extract are also immobilized on the surface. The
proteins of the cell extract and the substrates of an enzymatic
reaction are then incubated with a reaction mixture providing
conditions conducive to the occurrence of the enzymatic reaction.
The cellular repertoire of particular enzymatic activities can
thereby be assessed.
[0154] In a more specific embodiment, a plurality of different
substrates is immobilized on the surface of the solid support in a
well. In specific embodiments, a plurality of wells is present on
the microarray and each well contains the plurality of different
substrates. The proteins of a cellular extract are also immobilized
on the surface of the solid support in wells. Thus, different
enzymatic reactions can be tested simultaneously on the microarray.
In certain embodiments, the assay of the invention can be performed
with whole cells or preparations of plasma membranes. Thus, use of
several classes of substrates and reaction buffers can provide for
large-scale or exhaustive analysis of cellular activities. In
particular, one or several screens can form the basis of
identifying a "footprint" of the cell type or physiological state
of a cell, tissue, organ or system. For example, different cell
types (either morphological or functional) can be differentiated by
the pattern of cellular activities or expression determined by the
protein chip. This approach also can be used to determine, for
example, different stages of the cell cycle, disease states,
altered physiologic states (e.g., hypoxia), physiological state
before or after treatment (e.g., drug treatment), metabolic state,
stage of differentiation or development, response to environmental
stimuli (e.g., light, heat), cell-cell interactions, cell-specific
gene and/or protein expression, and disease-specific gene and/or
protein expression.
[0155] In a specific embodiment, compounds that modulate the
enzymatic activity of a protein or proteins on a chip can be
identified. For example, changes in the level of enzymatic activity
are detected and quantified by incubation of a compound or mixture
of compounds with an enzymatic reaction on the microarray, wherein
a signal is produced (e.g., from substrate that becomes fluorescent
upon enzymatic activity). Differences between the presence and
absence of the compound are noted. Furthermore, the differences in
effects of compounds on enzymatic activities of different proteins
are readily detected by comparing their relative effect on samples
within the protein chips and between chips.
[0156] In certain embodiments, the enzymatic activity detected
using a method of the invention is in part due to autocatalysis,
i.e., the enzyme acts on itself as well as on a substrate. A
nonlimiting example of autocatalysis is auto-phosphorylation.
[0157] In certain embodiments, immobilizing a substance and a
protein in proximity sufficient for the occurrence of an enzymatic
reaction between the substance and the protein induces the
catalytic activity of the protein. In certain embodiments,
immobilizing a substance and a protein in proximity sufficient for
the occurrence of an enzymatic reaction between the substance and
the protein induces the autocatalytic activity of the protein.
[0158] In certain embodiments, an enzymatic activity is enhanced by
immobilizing enzyme and substrate in proximity sufficient for the
occurrence of the enzymatic reaction. In a specific embodiment, the
activity is enhanced compared to the activity in solution.
[0159] Any enzyme known to the skilled artisan can be used with the
methods of the invention and with protein arrays of the invention.
Classes of enzymes include, but are not limited to,
Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, and
Ligases. Enzymes that can be used with the methods of the invention
and immobilization on the microarrays of the invention include but
are not limited to those shown in Table 1.
[0160] Identifying substrates of enzymes can also be conducted with
the methods of the present invention. For example, a wide variety
of different potential substrates are attached to the protein chip
and are assayed for their ability to act as a substrate for
particular enzyme(s) that is also immobilized to the surface of the
solid substrate.
[0161] In certain embodiments, candidate-substrates are identified
in a parallel experiment on the basis of a substrates' ability to
bind to the enzyme of interest. A substrate can be a cell,
protein-containing cellular material, protein, oligonucleotide,
polynucleotide, DNA, RNA, small molecule substrate, drug candidate,
receptor, antigen, steroid, phospholipid, antibody, immunoglobulin
domain, glutathione, maltose, nickel, dihydrotrypsin, or biotin.
After incubation of proteins on a chip with test molecules, the
candidate substrates can be identified by mass spectrometry (Lakey
et al., 1998, "Measuring protein-protein interactions", Curr Opin
Struct Biol. 8:119-23).
[0162] The identity of targets of a specific enzymatic activity can
be assayed by treating a protein chip with complex protein
mixtures, such as cell extracts, and determining protein activity,
wherein the complex protein mixture is also immobilized on the
surface of the solid support. For example, a protein chip
containing an array of different kinases can be contacted with a
cell extract from cells treated with a compound (e.g., a drug), and
assayed for kinase activity. In another example, a protein chip
containing an array of different kinases can be contacted with a
cell extract from cells at a particular stage of cell
differentiation (e.g., pluripotent) or from cells in a particular
metabolic state (e.g., mitotic), and assayed for kinase activity.
Proteins of the cell extract can be immobilized to the solid
support by methods as described above. The results obtained from
such assays, comparing for example, cells in the presence or
absence of a drug, or cells at several differentiation stages, or
cells in different metabolic states, can provide information
regarding the physiologic changes in the cells between the
different conditions.
[0163] Alternatively, the identity of targets of specific cellular
activities can be assayed by treating a protein chip, containing
many different proteins (e.g., a peptide library) immobilized to
the surface of the solid support of the protein chip, with a
complex protein mixture (e.g., such as a cell extract), and
assaying for modifications to the proteins on the chip, wherein the
protein mixture is also immobilized to the surface of the solid
support. For example, a protein chip containing an array of
different proteins can be contacted with a cell extract from cells
treated with a compound (e.g., a drug), and assayed for
oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase
activity. In more specific embodiments, kinase, protease,
glycosidase, actetylase, phosphatase, or other transferase activity
can for example be assayed. In another example, a protein chip
containing an array of different proteins can be contacted with a
cell extract from cells at a particular stage of cell
differentiation (e.g., pluripotent) or from cells in a particular
metabolic state (e.g., mitotic). The results obtained from such
assays, comparing for example, cells in the presence or absence of
a drug, or cells at several stages of differentiation, or cells in
different metabolic states, can provide information regarding the
physiologic effect on the cells under these conditions.
[0164] The activity of proteins exhibiting differences in function,
such as enzymatic activity, can be analyzed with the protein
methods of the present invention. For example, differences in
protein isoforms derived from different alleles are assayed for
their activities relative to one another.
[0165] The methods of the invention can be used for drug discovery,
analysis of the mode of action of a drug, drug specificity, and
prediction of drug toxicity. As many enzymes and substrates can be
tested at the same time, the methods of the invention are suitable
to determine profiles for different drugs. In certain embodiments,
such a profile relates to sensitivities of different enzymes to the
drug of interest. In other embodiments, such a profile relates to
effects of the drug of interest on the substrate specificity of
different enzymes. For example, the identity of proteins whose
activity is susceptible to a particular compound can be determined
by performing the assay of the invention in the presence and
absence of a compound. For a more detailed description of screening
assays using the methods of the invention, see section 5.6.
[0166] Moreover, the methods of the present invention can be used
to determine the presence of potential inhibitors, catalysts,
modulators, or enhancers of the enzymatic activity. In one example,
a cellular extract of a cell is added to an enzymatic assay of the
invention.
[0167] The protein chips of the invention can be used to determine
the effects of a drug on the modification of multiple targets by
complex protein mixtures, such as for example, whole cells, cell
extracts, or tissue homogenates. The net effect of a drug can be
analyzed by screening one or more protein chips with drug-treated
cells, tissues, or extracts, which then can provide a "signature"
for the drug-treated state, and when compared with the "signature"
of the untreated state, can be of predictive value with respect to,
for example, potency, toxicity, and side effects. Furthermore,
time-dependent effects of a drug can be assayed by, for example,
adding the drug to the cell, cell extract, tissue homogenate, or
whole organism, and applying the drug-treated cells or extracts to
a protein chip at various timepoints of the treatment.
[0168] In the subsections below, exemplary enzyme assays for use
with the invention are described. These examples are meant to
illustrate the present invention and are not intended to limit in
any way the scope of the present invention.
[0169] 5.2.1. Kinase Assay
[0170] In certain embodiments of the invention, the enzymatic
reaction to be performed with the methods of the invention is a
kinase reaction. In certain embodiments, a kinase is a tyrosine
kinase or a serine/threonine kinase. Exemplary kinases to be used
with the methods of the invention include, but not limited to, ABL,
ACK, AFK, AKT (e.g., AKT-1, AKT-2, and AKT-3), ALK, AMP-PK, ATM,
Aurora1, Aurora2, bARK1, bArk2, BLK, BMX, BTK, CAK, CaM kinase,
CDC2, CDK, CK, COT, CTD, DNA-PK, EGF-R, ErbB-1, ErbB-2, ErbB-3,
ErbB-4, ERK (e.g., ERK1, ERK2, ERK3, ERK4, ERK5, ERK6, ERK7),
ERT-PK, FAK, FGR (e.g., FGF1R, FGF2R), FLT (e.g., FLT-1, FLT-2,
FLT-3, FLT-4), FRK, FYN, GSK (e.g., GSK1, GSK2, GSK3-alpha,
GSK3-beta, GSK4, GSK5), G-protein coupled receptor kinases (GRKs),
HCK, HER2, HKII, JAK (e.g., JAK1, JAK2, JAK3, JAK4), JNK (e.g.,
JNK1, JNK2, JNK3), KDR, KIT, IGF-1 receptor, IKK-1, IKK-2, INSR
(insulin receptor), IRAK1, IRAK2, IRK, ITK, LCK, LOK, LYN, MAPK,
MAPKAPK-1, MAPKAPK-2, MEK, MET, MFPK, MHCK, MLCK, MLK3, NEU, NIK,
PDGF receptor alpha, PDGF receptor beta, PHK, PI-3 kinase, PKA,
PKB, PKC, PKG, PRK1, PYK2, p38 kinases, p135tyk2, p34cdc2, p42cdc2,
p42mapk, p44 mpk, RAF, RET, RIP, RIP-2, RK, RON, RS kinase, SRC,
SYK, S6K, TAK1, TEC, TIE1, TIE2, TRKA, TXK, TYK2, UL13, VEGFR1,
VEGFR2, YES, YRK, ZAP-70, and all subtypes of these kinases (see
e.g., Hardie and Hanks (1995) The Protein Kinase Facts Book, I and
II, Academic Press, San Diego, Calif.). A recent list of human
kinases can be found in Manning et al., 2002, Science
298:1912-1934. In certain embodiments of the invention, proteins to
be used with the methods of the invention and on the arrays of the
invention are proteins that have sequence homologies to a known
kinase.
[0171] In certain embodiments, the plurality of proteins and a
kinase substrate are immobilized on the surface of the solid
support. In certain, more specific embodiments, the plurality of
proteins consists of different known kinases. In certain
embodiments, a kinase and a plurality of different substrates are
immobilized on the surface of the solid support. In a specific
embodiment, at least one substrate is a known kinase substrate. The
substrates can be candidate substrates.
[0172] The kinase reaction can be visualized and quantified by any
method known to the skilled artisan. In specific embodiments, to
visualize the kinase reaction, ATP whose gamma-phosphate is
detectably labeled is added to the microarray in a reaction buffer.
The reaction buffer provides, in addition to ATP, reaction
conditions conducive to the kinase reaction. Reaction conditions
include, but are not limited to, pH, salt concentration,
concentration of Mg.sup.++, and detergent concentration. After
incubation in the reaction buffer, the microarray is washed to
remove any labeled ATP and the product is quantified via the
detectably labeled phosphate that has been transferred during the
kinase reaction from ATP to the substrate. The signal intensity is
proportional to the amount of labeled phosphate on the substrate
and thus to the activity of the kinase reaction.
[0173] The gamma phosphate of ATP can be detectably labeled by any
method known to the skilled artisan. In certain embodiments, the
gamma phosphate of ATP is labeled with radioactive phosphorus, such
as, but not limited to, .sup.32P or .sup.33P. .sup.35S-gamma-ATP
can also be used with the methods of the invention. If the
phosphate is labeled radioactively, the signal intensity can be
evaluated using autoradiography.
[0174] Without being bound by theory, some kinases act on a
substrate only in a particular molecular context. Such a molecular
context may, e.g., consist of certain scaffold proteins. In certain
embodiments of the invention, such scaffold proteins are provided
with the reaction buffer. In other embodiments, the scaffold
proteins are also immobilized on the surface of the solid
support.
[0175] In certain embodiments, a kinase reaction can be visualized
and quantified using antibodies that bind specifically to
phosphorylated proteins or peptides. Such antibodies include, but
are not limited to antibodies that bind to phospho-serine or
antibodies that bind to phospho-tyrosine. The antibody that binds
to the phosphorylated protein or peptide can be directly labeled
and detected by any method known to the skilled artisan. In other
embodiments, a secondary antibody is used to detect the antibody
that is bound to the phosphorylated protein or peptide. The more
active the kinase reaction is the more antibody will be bound and
the stronger the signal will be.
[0176] In certain embodiments, phosphorylation can be detected
using a molecule that binds to phosphate and that is linked to a
detectable label such as, but not limited to, a dye. In a specific
embodiment, a phosphorylated protein or peptide is detected using
Pro-Q Diamond stain from Molecular Probes.
[0177] 5.2.2. Phosphatase
[0178] Any phosphatase known to the skilled artisan can be used
with the methods of the present invention or for the manufacture of
the arrays of the present invention. Examples of protein tyrosine
phosphatases (PTPases) include, but are not limited to, PTP1B,
PTPMEG, PTP1c, Yop51, VH1, cdc25, CD45, HLAR, PTP18, HPTP.alpha.
and DPTP10D. See Zhang and Dixon, 1994, Adv. Enzym. 68: 1-36.
Examples of protein serine-threonine phosphatases include PP1,
PP2A, PP2B and PP2C. See Meth. Enzvm., Hunter & Sefton,
Academic press, New York, 201:389-398 (1991). In certain
embodiments, the proteins to be used with the methods of the
present invention or for the manufacture of the arrays of the
present invention have homologies with known phosphatases.
[0179] In certain embodiments, the plurality of proteins and a
phosphatase substrate are immobilized on the surface of the solid
support. In certain, more specific embodiments, the plurality of
proteins consists of different known phosphatases. In certain
embodiments, a phosphatase and a plurality of different substrates
are immobilized on the surface of the solid support. In a specific
embodiment, at least one substrate is a phosphatase substrate.
[0180] In certain embodiments, the phosphatase reaction is
performed by adding a phosphatase reaction buffer to phosphatase
and substrate on the surface of the solid support. The phosphatase
reaction buffer provides conditions conducive to the phosphatase
reaction.
[0181] Any method known to the skilled artisan can be employed to
detect and quantify phosphatase activity. In certain embodiments,
the phosphatase substrate is a phosphorylated peptide or protein,
wherein the phosphate is detectably labeled. Phosphatase activity
can be detected and quantified by virtue of a decrease in
detectable label on the substrate and thus on the surface of the
solid support. In other embodiments, the release of the detectably
labeled phosphate into the reaction buffer is determined.
[0182] The phosphate can be detectably labeled by any method known
to the skilled artisan. In certain embodiments, the phosphate is
radioactively labeled, such as, but not limited to, .sup.32P or
.sup.33P. .sup.35S-gamma-ATP can also be used. If the phosphate is
labeled radioactively, the signal intensity can be evaluated using
autoradiography.
[0183] In certain embodiments, a phosphatase reaction can be
visualized and quantified using antibodies that bind specifically
to phosphorylated proteins or peptides. Such antibodies include,
but are not limited to antibodies that bind to phospho-serine or
antibodies that bind to phospho-tyrosine. The antibody that binds
to the phosphorylated protein or peptide can be directly labeled
and detected by any method known to the skilled artisan. In other
embodiments, a secondary antibody is used to detect the antibody
that is bound to the phosphorylated protein or peptide. The more
active the phosphatase reaction is the less antibody will be bound
and the weaker the signal will be.
[0184] 5.2.3. Glycosidase
[0185] Any glycosidase known to the skilled artisan can be used
with the present invention. Glycosidases include, but are not
limited to, both endo- and exo-glucosidases which can cleave both
alpha- and beta-glycosidic bonds, for example amylase, maltase,
cellulase, endoxylanase, beta-glucanase, mannanase, or lysozyme,
and in addition galactosidase or beta-glucuronidases. In certain
embodiments, proteins to be used with the invention have homologies
to a known glycosidase.
[0186] In certain embodiments, the plurality of proteins and a
glycosidase substrate are immobilized on the surface of the solid
support. In certain, more specific embodiments, the plurality of
proteins consists of different known glycosidases. In certain
embodiments, a glycosidase and a plurality of different substrates
arc immobilized on the surface of the solid support. In a specific
embodiment, at least one substrate is a glycosidase substrate.
[0187] In certain embodiments, the glycosidase reaction is
performed by adding a glycosidase reaction buffer to glycosidase
and substrate on the surface of the solid support. The glycosidase
reaction buffer provides conditions conducive to the glycosidase
reaction.
[0188] Any method known to the skilled artisan can be employed to
detect and quantify glycosidase activity. In certain embodiments,
the glycosidase substrate is, e.g., a polysaccharide. Glycosidase
activity can be detected and quantified by virtue of a decrease in
detectable label on the substrate and thus on the surface of the
solid support. In other embodiments, the release of the detectably
labeled monomers of the polysaccharide into the reaction buffer is
measured.
[0189] The glycosidase substrate can be detectably labeled by any
method known to the skilled artisan. In certain embodiments, the
polysaccharide is radioactively labeled. In certain embodiments,
the polysaccharide is attached to the surface of the solid support
on one end and is detectably labeled on the other end. The decrease
of detectable label on the surface of the solid support is a
measure for the activity of the glycosidase activity.
[0190] 5.2.4. Protease
[0191] Any protease known to the skilled artisan can be used with
the present invention. Proteases include, but are not limited to,
Bromelain, Cathepsins, Chymotrypsin, Collagenase, Elastase,
Kallikrein, Papain, Pepsin, Plasmin, Renin, Streptokinase,
Subtilisin, Thermolysin, Thrombin, Trypsin, and Urokinase.
[0192] In certain embodiments, the plurality of proteins and a
protease substrates are immobilized on the surface of the solid
support. In certain, more specific embodiments, the plurality of
proteins consists of different known proteases. In certain
embodiments, a protease and a plurality of different protease
substrates are immobilized on the surface of the solid support.
[0193] In certain embodiments, the protease reaction is performed
by adding a protease reaction buffer to protease and substrate on
the surface of the solid support. The protease reaction buffer
provides conditions conducive to the glycosidase reaction.
[0194] Any method known to the skilled artisan can be employed to
detect and quantify protease activity. In certain embodiments, the
protease substrate is, e.g., a polypeptide or a protein. Protease
activity can be detected and quantified by virtue of a decrease in
detectable label on the substrate and thus on the surface of the
solid support. In other embodiments, the release of the detectably
labeled amino acids or peptides of the polypeptide into the
reaction buffer is measured. In certain other embodiments, FRET or
fluorescence polarization is used to detect and quantify a protease
reaction.
[0195] The protease substrate can be detectably labeled by any
method known to the skilled artisan. In certain embodiments, the
protein or polypeptide is radioactively labeled. In certain
embodiments, the protein or polypeptide is attached to the surface
of the solid support on one end and is detectably labeled on the
other end. The decrease of detectable label on the surface of the
solid support is a measure for the activity of the protease
activity.
[0196] In a specific embodiment, protease activity is assayed in
the following way. First, protein probes are prepared consisting of
various combinations of amino acids, with a C-terminal or
N-terminal mass spectroscopic label attached, with the only proviso
being that the molecular weight of the label should be sufficiently
large so that all labeled cleavage products of the protein can be
detected. The protein substrate is immobilized to the protein chip
and the proteases are immobilized to the protein chip in proximity
with each other sufficient to allow occurrence of the protease
reaction. After incubation at 37.degree. C. for an appropriate
period of time, and washing with acetonitrile and trifluoroacetic
acid, protease activity is measured by detecting the proteolytic
products using mass spectrometry. This assay provides information
regarding both the proteolytic activity and specificity of the
proteases attached to the protein chip.
[0197] Another rapid assay for protease activity analysis is to
attach proteins of known sequence to the chip. The substrate
proteins are fluorescently labeled at the end not attached to the
chip. Upon incubation with the protease(s) of interest, the
fluorescent label is lost upon proteolysis, such that decreases in
fluorescence indicate the presence and extent of protease activity.
This same type of assay can be carried out wherein the protein
substrates are attached to beads placed in the wells of the
chips.
[0198] 5.2.5 Nuclease
[0199] Nuclease activity can be assessed in the same manner as
described for protease activity (see section 5.2.4) except that
nucleic acid substrates are used instead of protein substrates. As
such, fluorescently tagged nucleic acid fragments that are released
by nuclease activity can be detected by fluorescence, or the
nucleic acid fragments can be detected directly by mass
spectrometry.
[0200] 5.3 Substrates and Cofactors
[0201] If substances are to be identified that serve as substrates
for a particular enzymatic reaction, any substance can be used with
the methods of the invention. In certain embodiments, combinatorial
libraries of molecules can be used as substances (see section 5.6.1
for libraries).
[0202] Substrates to be used as substrates of a particular
enzymatic reaction with the methods of the present invention can be
any substrate known to the skilled artisan of that particular
enzymatic reaction.
[0203] In certain embodiments, generic substrates for a particular
enzymatic reaction can be used. In other embodiments, the
substrates are specific for class of enzymes. In even other
embodiments, the substrates are specific for individual
enzymes.
[0204] In an illustrative embodiments, a substrate of an enzyme
that catalyzes the phosphorylation of tyrosine residues in a
protein or peptide is a protein or peptide with tyrosines. In
another illustrative embodiment, a substrate of an enzyme that
catalyzes the phosphorylation of serine and threonine residues in a
protein or peptide is a protein or peptide with serine and/or
threonine. A substrate for a dual specificity kinase has tyrosine
and/or serine and/or threonine. Certain kinases require a conserved
target motif in their substrate for phosphorylation. In certain
embodiments, such a conserved target motif is present in the
substrate. In a specific embodiment, a kinase substrate is, but is
not limited to, casein. In another specific embodiments, a mixture
of Myelin Basic Protein (MBP), histone and casein is used as
substrate. In another specific embodiments, a mixture of Myelin
Basic Protein (MBP), histone, casein and/or poly(Glu4Tyr) is used
as substrate.
[0205] Certain enzymes that use proteins or peptides as substrate
require the presence of a particular amino acid or amino acid motif
in their substrates for the enzymatic reaction to occur. Such sites
in a amino acid sequence that are used by a particular enzymatic
activity can be predicted using such databases as PROSITE.
[0206] Illustrative substrates for exemplary enzymes are listed in
Table 2.
2TABLE 2 Type of Reaction Substrate and Specific Class of Enzyme
Catalyzed Examples Reaction Catalyzed Oxidoreductases Oxidation-
Alcohol An alcohol + reduction dehydrogenase. NAD(+) <=>
reactions an aldehyde or ketone + NADH Transferases Transfer of
Nicotinamide N- S-adenosyl-L- functional groups methyltransferase.
methionine + nicotinamide <=> S-adenosyl-L- homocysteine +
1-methylnicotinamide Protein-tyrosine ATP + kinase. a protein
tyrosine <=> ADP + protein tyrosine phosphate Amino-acid N-
Acetyl-CoA + acetyltransferase. L-glutamate <=> CoA +
N-acetyl-L-glutamate Phosphorylase. {(1,4)-alpha-D- glucosyl}(N) +
phosphate <=> {(1,4)-alpha-D- glucosyl}(N-1) +
alpha-D-glucose 1- phosphate Aspartate L-aspartate +
aminotransferase. 2-oxoglutarate <=> oxaloacetate +
L-glutamate Hexokinase. ATP + D-hexose <=> ADP + D-hexose
6-phosphate Thiosulfate Thiosulfate + sulfurtransferase. cyanide
<=> sulfite + thiocyanate Hydrolases Hydrolysis Leucyl
Release of an N-terminal reactions aminopeptidase. amino acid,
Xaa-.vertline.-Xbb-, in which Xaa is preferably Leu, but may be
other amino acids including Pro although not Arg or Lys, and Xbb
may be Pro. Lyases Group Pyruvate A 2-oxo acid <=>
Elimination to decarboxylase. an aldehyde + form double CO(2) bonds
Isomerases Isomerization Phosphoglycerate 2-phospho-D-glycerate +
mutase. 2,3- diphosphoglycerate <=> 3-phospho-D-glycerate +
2,3- diphosphoglycerate Ligases Bond formation Tyrosine-tRNA ATP +
coupled with ATP ligase. L-tyrosine + hydrolysis tRNA(Tyr)
<=> AMP + diphosphate + L-tyrosyl-tRNA(Tyr)
[0207] 5.3.1 Cofactors
[0208] In certain embodiments of the invention, the enzymatic
reaction being assayed requires a cofactor. Cofactors can be added
to the reaction in the reaction mixture. Cofactors that can be used
with the methods of the invention include, but are not limited to,
5,10-methenyltetrahydrofol- ate, Ammonia, Ascorbate, ATP,
Bicarbonate, Bile salts, Biotin, Bis(molybdopterin guanine
dinucleotide)molybdenum cofactor, Cadmium, Calcium, Cobalamin,
Cobalt, Coenzyme F430, Coenzyme-A, Copper, Dipyrromethane,
Dithiothreitol, Divalent cation, FAD, Flavin, Flavoprotein, FMN,
Glutathione, Heme, Heme-thiolate, Iron, Iron(2+), Iron-molybdenum,
Iron-sulfur, Lipoyl group, Magnesium, Manganese, Metal ions,
Molybdenum, Molybdopterin, Monovalent cation, NAD, NAD(P)H, Nickel,
Potassium, PQQ, Protoheme IX, Pyridoxal-phosphate, Pyruvate,
Selenium, Siroheme, Sodium, Tetrahydropteridine, Thiamine
pyrophosphate, Topaquinone, Tryptophan tryptophylquinone (TTQ),
Tungsten, Vanadium, Zinc.
[0209] 5.4. Properties of the Protein Chips to be Used with the
Methods of the Invention
[0210] In various specific embodiments, the microarray of the
invention is a positionally addressable array comprising a
plurality of different proteins and a substance immobilized on the
surface of a solid support. In other embodiments, the microarray of
the invention is a positionally addressable array comprising a
plurality of different substances and an enzyme immobilized on the
surface of a solid support. In certain embodiments, the proteins
comprise a functional domain on a solid support. Each different
protein or substance is at a different position on the solid
support. In certain embodiments, the plurality of different
proteins or molecules consists of at least 50%, 75%, 90%, or 95% of
all expressed proteins with the same type of biological activity in
the genome of an organism. For example, such organism can be
eukaryotic or prokaryotic, and is preferably a mammal, a human or
non-human animal, primate, mouse, rat, cat, dog, horse, cow,
chicken, fungus such as yeast, Drosophila, C. elegans, etc. Such
type of biological activity of interest can be, but is not limited
to, enzymatic activity (e.g., kinase activity, protease activity,
phosphatase activity, glycosidase, acetylase activity, and other
chemical group transferring enzymatic activity), nucleic acid
binding, hormone binding, etc.
[0211] In certain embodiments, the plurality of different proteins
or substances is immobilized on the surface of the solid support at
a density of about 1 to 10, 5 to 20, 10 to 50, 30 to 100, about 30,
between 30 and 50, between 50 and 100, at least 100, between 100
and 1000, between 1000 and 10,000, between 10,000 and 100,000,
between 100,000 and 1,000,000, between 1,000,000 and 10,000,000,
between 10,000,000 and 25,000,000, at least 25,000,000, at least
10,000,000,000, or at least 10,000,000,000,000 different proteins
or substances, per cm.sup.2.
[0212] In certain embodiments, the plurality of different proteins
and a plurality of different substances are immobilized on the
surface of the solid support at a density of about 1 to 10, 5 to
20, 10 to 50, 30 to 100, about 30, between 30 and 50, between 50
and 100, at least 100, between 100 and 1000, between 1000 and
10,000, between 10,000 and 100,000, between 100,000 and 1,000,000,
between 1,000,000 and 10,000,000, between 10,000,000 and
25,000,000, at least 25,000,000, at least 10,000,000,000, or at
least 10,000,000,000,000 different proteins or substances,
respectively, per cm.sup.2.
[0213] The protein chips to be used with the present invention are
not limited in their physical dimensions and may have any
dimensions that are convenient. For the sake of compatibility with
current laboratory apparatus, protein chips the size of a standard
microscope slide or smaller are preferred. In certain embodiments,
protein chips are sized such that two chips fit on a microscope
slide. Also preferred are protein chips sized to fit into the
sample chamber of a mass spectrometer. Also preferred are
microtiter plates.
[0214] In certain embodiments, a substance and enzyme are
immobilized on the surface of a solid support within wells. In
certain embodiments, a plurality of different enzymes or different
substances is printed or coated on the surface of the solid support
such that each protein or substance of the microarray is in a
different well. In other embodiments, a plurality of different
enzymes or different substances is printed onto the surface of the
solid support such that each well harbors a plurality of different
proteins or substrates. The performance of the enzymatic reaction
on a solid support with wells has the advantage that different
reaction solutions can be added at the same time onto one solid
support (e.g., on one slide). Another advantage of wells over flat
surfaces is increased signal-to-noise ratios. Wells allow the use
of larger volumes of reaction solution in a denser configuration,
and therefore greater signal is possible. Furthermore, wells
decrease the rate of evaporation of the reaction solution from the
chip as compared to flat surface arrays, thus allowing longer
reaction times. Another advantage of wells over flat surfaces is
that the use of wells permit association studies using a specific
volume of reaction volume for each well on the chip, whereas the
use of flat surfaces usually involves indiscriminate probe
application across the whole substrate. The application of a
defined volume of reaction buffer can be important if a reactant
that is supplied in the reaction buffer is being depleted during
the course of the reaction. In such a scenario, the application of
a defined volume allows for more reproducible results.
[0215] In certain embodiments, if the microarrays to be used with
the methods of the invention and the microarrays of the invention
have wells, the wells in the protein chips may have any shape such
as rectangular, square, or oval, with circular being preferred. The
wells in the protein chips may have square or round bottoms,
V-shaped bottoms, or U-shaped bottoms. Square bottoms are slightly
preferred because the preferred reactive ion etch (RIE) process,
which is anisotropic, provides square-bottomed wells. The shape of
the well bottoms need not be uniform on a particular chip, but may
vary as required by the particular assay being carried out on the
chip.
[0216] The wells in the protein chips to be used with the methods
of the present invention may have any width-to-depth ratio, with
ratios of width-to-depth between about 10:1 and about 1:10 being
preferred. The wells in the protein chips may have any volume, with
wells having volumes of at least 1 pl, at least 10 pl, at least 100
pl, at least 1 nl, at least 10 nl, at least 100 nl, at least 1
.mu.l, at least 10 .mu.l, or at least 100 .mu.l. The wells in the
protein chips may have any volume, with wells having volumes of at
most 1 pl, at most 10 pl, at most 100 pl, at most 1 nl, at most 10
nl, at most 100 nl, at most 1 .mu.l, at most 10 .mu.l, or at most
100 .mu.l.
[0217] In certain embodiments, the wells are formed by placing a
gasket with openings on the surface of the solid support such that
the openings in the gasket form the wells. In certain, more
specific embodiments, an array has at least 1, 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 24, 50 or at least 100 wells. In certain, more
specific embodiments, an array has at most 1, 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 24, 50 or at least 100 wells.
[0218] The protein chips of the invention can have a wide variety
of density of wells/cm.sup.2. The density of wells is between about
1 well/cm.sup.2 and about 10,000,000,000,000 wells/cm.sup.2.
Densities of wells on protein chips cast from master molds of laser
milled Lucite are generally between 1 well/cm.sup.2 and 2,500
wells/cm.sup.2. Appropriate milling tools produce wells as small as
100 .mu.m in diameter and 100 .mu.m apart. Protein chips cast from
master mold etched by wet-chemical microlithographic techniques
have densities of wells generally between 50 wells/cm.sup.2 and
10,000,000,000 wells/cm.sup.2. Wet-chemical etching can produce
wells that are 10 .mu.m deep and 10 .mu.m apart, which in turn
produces wells that are less than 10 .mu.m in diameter. Protein
chips cast from master mold etched by RIE microlithographic
techniques have densities of wells generally between 100
wells/cm.sup.2 and 25,000,000 wells/cm.sup.2. RIE in combination
with optical lithography can produce wells that are 500 nm in
diameter and 500 nm apart. Use of electron beam lithography in
combination with RIE can produce wells 50 nm in diameter and 50 nm
apart. Wells of this size and with equivalent spacing produces
protein chips with densities of wells 10,000,000,000,000
wells/cm.sup.2. Preferably, RIE is used to produce wells of 20
.mu.m in diameter and 20 .mu.m apart. Wells of this size that are
equivalently spaced will result in densities of 25,000,000
wells/cm.sup.2.
[0219] In a specific embodiment, the microarray is prepared on a
slide with 8 to 10 wells per slide, wherein the plurality of
proteins is present in each well on the slide. In another
embodiment, microarray is prepared on a slide with 8 to 10 wells
per slide, wherein the plurality of substrates is present in each
well on the slide.
[0220] In one embodiment, the array comprises a plurality of wells
on the surface of a solid support wherein the density of wells is
at least 1 well/cm.sup.2, at least 10 wells/cm.sup.2, 100
wells/cm.sup.2. In another embodiment, said density of wells is
between 1000 and 1,000 wells/cm.sup.2. In another embodiment, said
density of wells is between 1000 and 100,000 wells/cm.sup.2. In
another embodiment, said density of wells is between 10,000 and
100,000 wells/cm.sup.2. In yet another embodiment, said density of
wells is between 100,000 and 1,000,000 wells/cm.sup.2. In yet
another embodiment, said density of wells is between between is
10,000,000 and 10,000,000 wells/cm.sup.2. In yet another
embodiment, said density of of wells is at least 25,000,000
wells/cm.sup.2. In yet another embodiment, said density of wells is
at least 10,000,000,000 wells/cm.sup.2. In yet another embodiment,
said density of wells is at least 10,000,000,000,000
wells/cm.sup.2.
[0221] The placement of a proteins or a substance can be
accomplished by using any dispensing means, such as bubble jet or
ink jet printer heads. A micropipette dispenser can also be used.
The placement of proteins or probes can either be conducted
manually or the process can be automated through the use of a
computer connected to a machine.
[0222] The present invention contemplates a variety of solid
supports cast from a microfabricated mold, some of which are
disclosed, for example, in international patent application
publication WO 01/83827, published Nov. 8, 2001, which is
incorporated herein by reference in its entirety.
[0223] In certain embodiments, the plurality of proteins comprises
all proteins that are encoded by at least 1%, 2%, 3%, 4%, 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the known
genes in a single species. In certain embodiments, the plurality of
substances comprises all proteins that are encoded by at least 1%,
2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
or 99% of the known genes in a single species.
[0224] In certain embodiments, the plurality of proteins comprises
at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, or 99% of all proteins expressed in a single
species, wherein protein isoforms and splice variants are counted
as a single protein. In certain embodiments, the plurality of
substances comprises at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all proteins expressed
in a single species, wherein protein isoforms and splice variants
are counted as a single protein.
[0225] In another embodiment, the plurality of proteins comprises
at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 500, 1000,
1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000,
100, 000, 500,000 or 1,000,000 protein(s) expressed in a single
species. In another embodiment, the plurality of substances
comprises at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200,
500, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000,
9000, 10,000, 100, 000, 500,000 or 1,000,000 protein(s) expressed
in a single species.
[0226] In yet another embodiment, the plurality of proteins
comprises proteins that are encoded by 1, 2, 3, 4, 5, 10, 20, 30,
40, 50, 100, 200, 500, 1000, 5000, 10000, 20000, 30000, 40000, or
50000 different known genes in a single species. In yet another
embodiment, the plurality of substances comprises proteins that are
encoded by 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 500, 1000,
5000, 10000, 20000, 30000, 40000, or 50000 different known genes in
a single species.
[0227] 5.5. Methods for Making and Purifying Proteins
[0228] Any method known to the skilled artisan can be used to make
and to purify the proteins to be used with the methods of the
invention and for the preparation of the microarrays of the
invention. In certain embodiments, the substrate is also a
proteinaceous molecule, such as a protein, a polypeptide or a
peptide and can be prepared and purified as described in this
section.
[0229] Proteins to be used with the methods of the invention and
for the preparation of the microarrays of the invention can be
fusion proteins, in which a defined domain is attached to one of a
variety of natural proteins, or can be intact non-fusion proteins.
In certain embodiments, if the substrate is a protein or a peptide,
a substrate to be used with the methods of the invention and for
the preparation of the microarrays of the invention can be fusion
protein, in which a defined domain is attached to the substrate, or
can be intact non-fusion substrate.
[0230] The present invention also relates to methods for making and
isolating viral, prokaryotic or eukaryotic proteins in a readily
scalable format, amenable to high-throughput analysis. Preferred
methods include synthesizing and purifying proteins in an array
format compatible with automation technologies. Accordingly, in one
embodiment, the invention provides a method for making and
isolating eukaryotic proteins comprising the steps of growing a
eukaryotic cell transformed with a vector having a heterologous
sequence operatively linked to a regulatory sequence, contacting
the regulatory sequence with an inducer that enhances expression of
a protein encoded by the heterologous sequence, lysing the cell,
contacting the protein with a binding agent such that a complex
between the protein and binding agent is formed, isolating the
complex from cellular debris, and isolating the protein from the
complex, wherein each step is conducted, e.g., in a 96-well
format.
[0231] In certain embodiments, the plurality of proteins comprises
at least one protein with a first tag and a second tag. In yet
another embodiment, the plurality of substrates comprises at least
one substrate with a first tag and a second tag.
[0232] In one embodiment, each step in the synthesis and
purification procedures is conducted in an array amenable to rapid
automation. Such arrays can comprise a plurality of wells on the
surface of a solid support wherein the density of wells is at least
10, 20, 30, 40, 50, 100, 1000, 10,000, 100,000, or 1,000,000
wells/cm.sup.2, for example. Alternatively, such arrays comprise a
plurality of sites on the surface of a solid support, wherein the
density of sites is at least 10, 20, 30, 40, 50, 100, 1000, 10,000,
100,000, or 1,000,000 sites/cm.sup.2, for example.
[0233] In a particular embodiment, proteins and/or substrates are
made and purified in a 96-array format (i.e., each site on the
solid support where processing occurs is one of 96 sites), e.g., in
a 96-well microtiter plate. In a preferred embodiment, the surface
of the microtiter plate that is used for the production of the
proteins and/or substrates does not bind proteins (e.g., a
non-protein-binding microtiter plate).
[0234] In certain embodiments, proteins and/or substrates are
synthesized by in vitro translation according to methods commonly
known in the art.
[0235] Any expression construct having an inducible promoter to
drive protein synthesis and/or the synthesis of a substrate (if the
substrate(s) is a protein or peptide) can be used in accordance
with the methods of the invention. Preferably, the expression
construct is tailored to the cell type to be used for
transformation. Compatibility between expression constructs and
host cells are known in the art, and use of variants thereof are
also encompassed by the invention.
[0236] Any host cell that can be grown in culture can be used to
synthesize the proteins and/or substrates of interest. Preferably,
host cells are used that can overproduce a protein and/or a
substrate of interest, resulting in proper synthesis, folding, and
posttranslational modification of the protein. Preferably, such
protein processing forms epitopes, active sites, binding sites,
etc. useful for the activity of an enzyme or the suitability as a
substrate. Posttranslational modification is relevant if the
enzyme's activity is affected by posttranslational modification of
the enzyme. Posttranslational modification is also relevant if the
substrates ability to serve as a substrate for the enzymatic
reaction of interest is affected by the posttranslational
modification of the substrate. In a specific embodiment,
phosphorylation of a protein is required for the enzymatic activity
of the protein. In such a case the protein should be expressed in a
system that promotes the phosphorylation of the protein at the
appropriate site. In a specific embodiment, phosphorylation or
glycosylation of a substrate is required for the substrate to
modified by the enzymatic reaction of interest. In such a case the
substrate should be synthesized in a system that promotes the
phosphorylation or glycosylation of the substrate at the
appropriate site.
[0237] Accordingly, a eukaryotic cell (e.g., yeast, human cells) is
preferably used to synthesize eukaryotic proteins or substrates of
eukaryotic enzymes. Further, a eukaryotic cell amenable to stable
transformation, and having selectable markers for identification
and isolation of cells containing transformants of interest, is
preferred. Alternatively, a eukaryotic host cell deficient in a
gene product is transformed with an expression construct
complementing the deficiency. Cells useful for expression of
engineered viral, prokaryotic or eukaryotic proteins are known in
the art, and variants of such cells can be appreciated by one of
ordinary skill in the art.
[0238] For example, the InsectSelect system from Invitrogen
(Carlsbad, Calif., catalog no. K800-01), a non-lytic, single-vector
insect expression system that simplifies expression of high-quality
proteins and eliminates the need to generate and amplify virus
stocks, can be used. A preferred vector in this system is
pIB/V5-His TOPO TA vector (catalog no. K890-20). Polymerase chain
reaction ("PCR") products can be cloned directly into this vector,
using the protocols described by the manufacturer, and the proteins
can be expressed with N-terminal histidine tags useful for
purifying the expressed protein.
[0239] Another eukaryotic expression system in insect cells, the
BAC-TO-BAC.TM. system (LIFETECH.TM., Rockville, Md.), can also be
used. Rather than using homologous recombination, the
BAC-TO-BAC.TM. system generates recombinant baculovirus by relying
on site-specific transposition in E. coli. Gene expression is
driven by the highly active polyhedrin promoter, and therefore can
represent up to 25% of the cellular protein in infected insect
cells.
[0240] In a particular embodiment, yeast cultures are used to
synthesize eukaryotic fusion proteins. Fresh cultures are
preferably used for efficient induction of protein synthesis,
especially when conducted in small volumes of media. Also, care is
preferably taken to prevent overgrowth of the yeast cultures. In
addition, yeast cultures of about 3 ml or less are preferable to
yield sufficient protein for purification. To improve aeration of
the cultures, the total volume can be divided into several smaller
volumes (e.g., four 0.75 ml cultures can be prepared to produce a
total volume of 3 ml).
[0241] Cells are then contacted with an inducer, and harvested. The
nature of the inducer depends on the expression system used. The
nature of the inducer particularly depends on the promoter used. In
certain embodiments, the expression system used for the preparation
of the proteins and/or substrates is an inducible expression
system. Any inducible expression system known to the skilled
artisan can be used with the methods of the invention and for the
preparation of the microarrays of the invention. Examples of
inducers include, but are not limited to, galactose,
enhancer-binding proteins, and other transcription factors. In one
embodiment, galactose is contacted with a regulatory sequence
comprising a galactose-inducible GAL1 promoter.
[0242] Induced cells are washed with cold (i.e., 4.degree. C. to
about 15.degree. C.) water to stop further growth of the cells, and
then washed with cold (i.e., 4.degree. C. to about 15.degree. C.)
lysis buffer to remove the culture medium and to precondition the
induced cells for protein purification, respectively. Before
protein purification, the induced cells can be stored frozen to
protect the proteins from degradation. In a specific embodiment,
the induced cells are stored in a semi-dried state at -80.degree.
C. to prevent or inhibit protein degradation.
[0243] Cells can be transferred from one array to another using any
suitable mechanical device. For example, arrays containing growth
media can be inoculated with the cells of interest using an
automatic handling system (e.g., automatic pipette). In a
particular embodiment, 96-well arrays containing a growth medium
comprising agar can be inoculated with yeast cells using a
96-pronger. Similarly, transfer of liquids (e.g., reagents) from
one array to another can be accomplished using an automated
liquid-handling device (e.g., Q-FILL.TM., Genetix, UK).
[0244] Although proteins can be harvested from cells at any point
in the cell cycle, cells are preferably isolated during logarithmic
phase when protein synthesis is enhanced. For example, yeast cells
can be harvested between OD.sub.600=0.3 and OD.sub.600=1.5,
preferably between OD.sub.600=0.5 and OD.sub.600=1.5. In a
particular embodiment, proteins are harvested from the cells at a
point after mid-log phase. Harvested cells can be stored frozen for
future manipulation.
[0245] The harvested cells can be lysed by a variety of methods
known in the art, including mechanical force, enzymatic digestion,
and chemical treatment. The method of lysis should be suited to the
type of host cell. For example, a lysis buffer containing fresh
protease inhibitors is added to yeast cells, along with an agent
that disrupts the cell wall (e.g., sand, glass beads, zirconia
beads), after which the mixture is shaken violently using a shaker
(e.g., vortexer, paint shaker).
[0246] In a specific embodiment, zirconia beads are contacted with
the yeast cells, and the cells lysed by mechanical disruption by
vortexing. In a further embodiment, lysing of the yeast cells in a
high-density array format is accomplished using a paint shaker. The
paint shaker has a platform that can firmly hold at least eighteen
96-well boxes in three layers, thereby allowing for high-throughput
processing of the cultures. Further the paint shaker violently
agitates the cultures, even before they are completely thawed,
resulting in efficient disruption of the cells while minimizing
protein degradation. In fact, as determined by microscopic
observation, greater than 90% of the yeast cells can be lysed in
under two minutes of shaking.
[0247] The resulting cellular debris can be separated from the
protein and/or substrate of interest by centrifugation.
Additionally, to increase purity of the protein sample in a
high-throughput fashion, the protein-enriched supernatant can be
filtered, preferably using a filter on a non-protein-binding solid
support. To separate the soluble fraction, which contains the
proteins of interest, from the insoluble fraction, use of a filter
plate is highly preferred to reduce or avoid protein degradation.
Further, these steps preferably are repeated on the fraction
containing the cellular debris to increase the yield of
protein.
[0248] Proteins and/or substrates can then be purified from the
protein-enriched supernatant using a variety of affinity
purification methods known in the art. Affinity tags useful for
affinity purification of fusion proteins by contacting the fusion
protein preparation with the binding partner to the affinity tag,
include, but are not limited to, calmodulin,
trypsin/anhydrotrypsin, glutathione, immunoglobulin domains,
maltose, nickel, or biotin and its derivatives, which bind to
calmodulin-binding protein, bovine pancreatic trypsin inhibitor,
glutathione-S-transferase ("GST tag"), antigen or Protein A,
maltose binding protein, poly-histidine ("His tag"), and
avidin/streptavidin, respectively. Other affinity tags can be, for
example, myc or FLAG. Fusion proteins can be affinity purified
using an appropriate binding compound (i.e., binding partner such
as a glutathione bead), and isolated by, for example, capturing the
complex containing bound proteins on a non-protein-binding filter.
Placing one affinity tag on one end of the protein (e.g., the
carboxy-terminal end), and a second affinity tag on the other end
of the protein (e.g., the amino-terminal end) can aid in purifying
full-length proteins.
[0249] In certain embodiments, a protein and/or a substance is
expressed as a fusion protein with a chitin binding domain. In
other embodiments, a protein and/or a substance is expressed as a
fusion protein with a chitin binding domain and an intein. In a
more specific embodiment, the proteins and/or substrates are
expressed using the IMPACT.TM.-CN system from New England Biolabs
Inc.
[0250] In a particular embodiment, the fusion proteins have GST
tags and are affinity purified by contacting the proteins with
glutathione beads. In further embodiment, the glutathione beads,
with fusion proteins attached, can be washed in a 96-well box
without using a filter plate to ease handling of the samples and
prevent cross contamination of the samples.
[0251] In addition, fusion proteins can be eluted from the binding
compound (e.g., glutathione bead) with elution buffer to provide a
desired protein concentration.
[0252] For purified proteins and/or substrates that will eventually
be printed or coated onto the surface of the solid support, such
as, but not limited to, a microscope slide, the glutathione beads
are separated from the purified proteins and/or substrates.
Preferably, all of the glutathione beads are removed to avoid
blocking of the microarrays pins used to spot the purified proteins
onto a solid support. In a preferred embodiment, the glutathione
beads are separated from the purified proteins using a filter
plate, preferably comprising a non-protein-binding solid support.
Filtration of the eluate containing the purified proteins should
result in greater than 90% recovery of the proteins.
[0253] The elution buffer preferably comprises a liquid of high
viscosity such as, for example, 15% to 50% glycerol, preferably
about 25% glycerol. The glycerol solution stabilizes the proteins
and/or substrates in solution, and prevents dehydration of the
protein solution during the printing step using a microarrayer.
[0254] Purified proteins and/or substrates are preferably stored in
a medium that stabilizes the proteins and prevents dessication of
the sample. For example, purified proteins can be stored in a
liquid of high viscosity such as, for example, 15% to 50% glycerol,
preferably in about 25% glycerol. It is preferred to aliquot
samples containing the purified proteins, so as to avoid loss of
protein activity caused by freeze/thaw cycles.
[0255] The skilled artisan can appreciate that the purification
protocol can be adjusted to control the level of protein purity
desired. In some instances, isolation of molecules that associate
with the protein of interest is desired. For example, dimers,
trimers, or higher order homotypic or heterotypic complexes
comprising an overproduced protein of interest can be isolated
using the purification methods provided herein, or modifications
thereof. Furthermore, associated molecules can be individually
isolated and identified using methods known in the art (e.g., mass
spectroscopy).
[0256] In certain embodiments, an enzyme to be used with the
invention is composed of two or more proteins in a complex. In such
a case, any method known to the skilled artisan can be used to
provide the complex for use with the methods of the invention. In a
specific embodiment, the proteins of the complex are co-expressed
and the proteins are purified as a complex. In other embodiments,
the proteins of the complex are expressed as a fusion protein that
comprises all proteins of the complex. The fusion protein may or
may not comprise linker peptides between the individual proteins of
the complex. In other embodiments, the proteins of the complex are
expressed, purified and subsequently incubated under conditions
that allow formation of the complex. In certain embodiments, the
proteins of the complex are assembled on the surface of the solid
support before they become immobilized. In even other embodiments,
the individual proteins of an enzymatic complex of interest are
printed on top of each other on the surface of the solid support.
Without being bound by theory, once the proteins of the complex are
immobilized on the surface the close physical proximity of the
proteins of the complex to each other allows for the enzymatic
reaction to take place even though the complex is not
assembled.
[0257] The protein and/or substrate can be purified prior to
placement on the protein chip or can be purified during placement
on the chip via the use of reagents that bind to particular
proteins, which have been previously placed on the protein chip.
Partially purified protein-containing cellular material or cells
can be obtained by standard techniques (e.g., affinity or column
chromatography) or by isolating centrifugation samples (e.g., P1 or
P2 fractions).
[0258] 5.5.1. Tagged Proteins
[0259] In certain embodiments, the proteins and/or substances to be
used with the methods of the invention or for the preparation of
the microarrays of the invention comprise a first tag and a second
tag. The advantages of using double-tagged proteins include the
ability to obtain highly purified proteins, as well as providing a
streamlined manner of purifying proteins from cellular debris and
attaching the proteins to a solid support. In a particular
embodiment, the first tag is a glutathione-S-transferase tag ("GST
tag") and the second tag is a poly-histidine tag ("His tag"). In a
specific embodiment, the poly-histidine tag consists of six
histidines (Hisx6). In other embodiments, the poly-histidine tag
consists of 4, 5, 7, 8, 9, 10, 11, or 12 histidines. In a further
embodiment, the GST tag and the His tag are attached to the
amino-terminal end of the protein or the substrate. Alternatively,
the GST tag and the His tag are attached to the carboxy-terminal
end of the protein or substrate.
[0260] In a preferred embodiment, a protein and/or a substrate is
expressed using the IPACT.TM.-CN system from New England Biolabs
Inc.
[0261] In yet another embodiment, the GST tag is attached to the
amino-terminal end of the protein or substrate. In a further
embodiment, the His tag is attached to the carboxy-terminal end of
the protein or substrate. In yet another embodiment, the His tag is
attached to the amino-terminal end of the protein or substrate. In
a further embodiment, the GST tag is attached to the
carboxy-terminal end of the protein or substrate.
[0262] In yet another embodiment, the protein or substrate
comprises a GST tag and a His tag, and neither the GST tag nor the
His tag is located at the amino-terminal or carboxy-terminal end of
the protein. In a specific embodiment, the GST tag and His tag are
located within the coding region of the protein or substrate of
interest; preferably in a region of the protein not affecting the
enzymatic activity of interest and preferably in a region of the
substrate not affecting the suitability of the substrate to be
modified by the enzymatic reaction of interest.
[0263] In one embodiment, the first tag is used to purify a fusion
protein. In another embodiment, the second tag is used to attach a
fusion protein to a solid support. In a specific further
embodiment, the first tag is a GST tag and the second tag is a His
tag.
[0264] A binding agent that can be used to purify a protein or a
substrate can be, but is not limited to, a glutathione bead, a
nickel-coated solid support, and an antibody. In one embodiment,
the complex comprises a fusion protein having a GST tag bound to a
glutathione bead. In another embodiment, the complex comprises a
fusion protein having a His tag bound to a nickel-coated solid
support. In yet another embodiment, the complex comprises the
protein of interest bound to an antibody and, optionally, a
secondary antibody.
[0265] 5.6. Screening Assays
[0266] The methods of the invention and the protein microarrays of
the invention can be used to identify molecules that modify the
activity or substrate-specificity of an enzyme or a class of
enzymes. In particular, the methods of the invention and the
protein microarrays of the invention can be used to identify a
molecule with a particular profile of activity, i.e., the molecule
modifies certain enzymes and does not affect the activity of other
enzymes. Such an assay is particularly useful to identify compounds
that are modulators of a desired specificity, wherein the compound
with the highest specificity modifies the activity of only one
specific enzyme and a compound with a lower specificity modifies
the activity of a subclass of enzymes. Modulators of an enzymatic
activity can be activators of the enzymatic activity, inhibitors of
the enzymatic activity or modulators of the enzyme's substrate
specificity. An inhibitor of an enzymatic reaction can inhibit the
enzyme reversably, irreversably, competitively, or
non-competitively.
[0267] In certain embodiments, a screening assay of the invention
is performed by conducting the enzyme assay on a microarray as
described in section 5.2, wherein the reaction is performed in the
presence and the absence of a molecule that is to be tested for its
effect on the enzymatic reaction. The effect of the test molecule
on the enzymatic reaction can be determined by comparing the
activity in the presence of the test molecule with the activity in
the absence of the test compound. In certain embodiments, if the
assay is performed in wells, several molecules can be tested
simultaneously on the same microarray. In certain embodiments, if
the assay is performed in wells, different concentrations of a
molecule can be tested simultaneously on the same microarray.
[0268] In certain embodiments, a molecule is tested for its effect
on the activity of an enzymatic reaction, wherein a plurality of
different enzymes and a substrate are immobilized to the surface of
the solid support. In a specific embodiment, the substrate may be a
known substrate of at least one of the enzymes. This is the
preferred embodiment, if the molecule is tested for an effect on
enzyme activity. If substrate specificity of an enzyme of interest
is to be tested, the preferred embodiment is to perform the assay
on a microarray wherein a plurality of different substrates and the
enzyme of interest are immobilized on the surface of a solid
support.
[0269] In other embodiments, the methods of the invention and the
microarrays of the invention can be used to identify a substrate
that is utilized by an enzyme of interest, a subclass of interest
or a class of enzymes.
[0270] In certain embodiments, the methods of the invention are
used to determine a profile of enzymatic activities of a cell in a
particular state of development or proliferation or of a cell of a
particular cell type. In a specific embodiment, the methods of the
invention are used to determine a profile of enzymatic activities
of a cell that is pre-neoplastic, neoplastic or cancerous in
comparison to a non-neoplastic or non-cancerous, respectively,
cell. In a specific embodiment, a cell extract of a cell type of
interest is immobilized on the surface of a solid support and a
plurality of different substrates is also immobilized on the
surface. In a more specific embodiment, the cell extract is size
fractionated and the different fractions are used with the methods
of the invention to enrich for the enzymes of interest in the cell
extract. In an even more specific embodiment, at least one enzyme
is isolated from a cell of interest and tested for its activity
using the methods of the invention.
[0271] In certain embodiments, kinetic properties of a known
inhibitor of a certain enzymes are assessed using the methods of
the invention. In certain, more specific embodiments, at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50 or at
least 100 copies of the plurality of different proteins are
immobilized on the surface of a solid support at different
positions of the microarray. The different proteins of at least 1
copy of the plurality of different proteins on the microarray are
in proximity with a substance sufficient for the occurrence of an
enzymatic reaction between the protein of the plurality of
different proteins and the substance. The different copies of the
plurality of different proteins can then incubated with different
reaction mixtures. The different reaction mixtures can each contain
a different test molecule that is to be tested for its effect on
the enzymatic reaction being assayed. In other embodiments, the
different reaction mixtures can each contain a different
concentration of a test molecule or known inhibitor or activator of
the enzymatic reaction. In certain embodiments, the different
copies of the plurality of different proteins are in different
wells on the solid support.
[0272] In certain, more specific embodiments, a at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50 or at least
100 copies of a plurality of different substances are immobilized
on the surface of a solid support at different positions of the
microarray. The different substances of at least 1 copy of the
plurality of different substances on the microarray are in
proximity with a protein sufficient for the occurrence of an
enzymatic reaction between the substances of the plurality of
different substance and the protein. The different copies of the
plurality of different substances can then incubated with different
reaction mixtures. The different reaction mixtures can each contain
a different test molecule that is to be tested for its effect on
the enzymatic reaction being assayed. In other embodiments, the
different reaction mixtures can each contain a different
concentration of a test molecule or known inhibitor or activator of
the enzymatic reaction. In certain embodiments, the different
copies of the plurality of different substances are in different
wells on the solid support.
[0273] 5.6.1 Libraries of Molecules
[0274] Any molecule known to the skilled artisan can be used with
the methods of the invention to test the molecule's effect on the
enzymatic reaction being assayed. In other embodiments, any
molecule can be used as a candidate substrate with the methods of
the invention. In certain embodiments, a library of different
molecules is used with the methods of the invention. Each member of
a library can be used as a test molecule to test its effect on the
enzymatic reaction or as a substance to test its suitability as a
substrate for the reaction being assayed. In certain embodiments,
the members of the library are tested individually. In other
embodiments, the members of a library are tested initially in
pools. The size of a pool can be at least 2, 10, 50, 100, 500,
1000, 5,000, or at least 10,000 different molecules. Once a
positive pool is identified, fractions of the pool can be tested or
the individual members of the pool of molecules are tested.
[0275] Libraries can contain a variety of types of molecules.
Examples of libraries that can be screened in accordance with the
methods of the invention include, but are not limited to, peptoids;
random biooligomers; diversomers such as hydantoins,
benzodiazepines and dipeptides; vinylogous polypeptides;
nonpeptidal peptidomimetics; oligocarbamates; peptidyl
phosphonates; peptide nucleic acid libraries; antibody libraries;
carbohydrate libraries; and small molecule libraries (preferably,
small organic molecule libraries). In some embodiments, the
molecules in the libraries screened are nucleic acid or peptide
molecules. In a non-limiting example, peptide molecules can exist
in a phage display library. In other embodiments, the types of
compounds include, but are not limited to, peptide analogs
including peptides comprising non-naturally occurring amino acids,
e.g., D-amino acids, phosphorous analogs of amino acids, such as
.alpha.-amino phosphoric acids and .alpha.-amino phosphoric acids,
or amino acids having non-peptide linkages, nucleic acid analogs
such as phosphorothioates and PNAs, hormones, antigens, synthetic
or naturally occurring drugs, opiates, dopamine, serotonin,
catecholamines, thrombin, acetylcholine, prostaglandins, organic
molecules, pheromones, adenosine, sucrose, glucose, lactose and
galactose. Libraries of polypeptides or proteins can also be used
in the assays of the invention.
[0276] In certain embodiments, combinatorial libraries of small
organic molecules including, but not limited to, benzodiazepines,
isoprenoids, thiazolidinones, metathiazanones, pyrrolidines,
morpholino compounds, and benzodiazepines. In another embodiment,
the combinatorial libraries comprise peptoids; random
bio-oligomers; benzodiazepines; diversomers such as hydantoins,
benzodiazepines and dipeptides; vinylogous polypeptides;
nonpeptidal peptidomimetics; oligocarbamates; peptidyl
phosphonates; peptide nucleic acid libraries; antibody libraries;
or carbohydrate libraries can be used with the methods of the
invention. Combinatorial libraries are themselves commercially
available (see, e.g., ComGenex, Princeton, N.J.; Asinex, Moscow,
Ru, Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, Russia; 3D
Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.;
etc.).
[0277] In a preferred embodiment, the library is preselected so
that molecules of the library are of the general type of molecules
that are being used in the enzymatic reaction of interest.
[0278] The combinatorial molecule library for use in accordance
with the methods of the present invention may be synthesized. There
is a great interest in synthetic methods directed toward the
creation of large collections of small organic compounds, or
libraries, which could be screened for pharmacological, biological
or other activity. The synthetic methods applied to create vast
combinatorial libraries are performed in solution or in the solid
phase, i.e., on a solid support. Solid-phase synthesis makes it
easier to conduct multi-step reactions and to drive reactions to
completion with high yields because excess reagents can be easily
added and washed away after each reaction step. Solid-phase
combinatorial synthesis also tends to improve isolation,
purification and screening. However, the more traditional solution
phase chemistry supports a wider variety of organic reactions than
solid-phase chemistry.
[0279] Combinatorial molecule libraries to be used in accordance
with the methods of the present invention may be synthesized using
the apparatus described in U.S. Pat. No. 6,190,619 to Kilcoin et
al., which is hereby incorporated by reference in its entirety.
U.S. Pat. No. 6,190,619 discloses a synthesis apparatus capable of
holding a plurality of reaction vessels for parallel synthesis of
multiple discrete compounds or for combinatorial libraries of
compounds.
[0280] In one embodiment, the combinatorial molecule library can be
synthesized in solution. The method disclosed in U.S. Pat. No.
6,194,612 to Boger et al., which is hereby incorporated by
reference in its entirety, features compounds useful as templates
for solution phase synthesis of combinatorial libraries. The
template is designed to permit reaction products to be easily
purified from unreacted reactants using liquid/liquid or
solid/liquid extractions. The compounds produced by combinatorial
synthesis using the template will preferably be small organic
molecules. Some compounds in the library may mimic the effects of
non-peptides or peptides. In contrast to solid phase synthesize of
combinatorial compound libraries, liquid phase synthesis does not
require the use of specialized protocols for monitoring the
individual steps of a multistep solid phase synthesis (Egner et
al., 1995, J. Org. Chem. 60:2652; Anderson et al., 1995, J. Org.
Chem. 60:2650; Fitch et al., 1994, J. Org. Chem. 59:7955; Look et
al., 1994, J. Org. Chem. 49:7588; Metzger et al., 1993, Angew.
Chem., Int. Ed. Engl. 32:894; Youngquist et al., 1994, Rapid
Commun. Mass Spect. 8:77; Chu et al., 1995, J. Am. Chem. Soc.
117:5419; Brummel et al., 1994, Science 264:399; and Stevanovic et
al., 1993, Bioorg. Med. Chem. Lett. 3:431).
[0281] Combinatorial molecule libraries useful for the methods of
the present invention can be synthesized on solid supports. In one
embodiment, a split synthesis method, a protocol of separating and
mixing solid supports during the synthesis, is used to synthesize a
library of compounds on solid supports (see e.g., Lam et al., 1997,
Chem. Rev. 97:41-448; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci.
USA 90:10922-10926 and references cited therein). Each solid
support in the final library has substantially one type of compound
attached to its surface. Other methods for synthesizing
combinatorial libraries on solid supports, wherein one product is
attached to each support, will be known to those of skill in the
art (see, e.g., Nefzi et al., 1997, Chem. Rev. 97:449-472).
[0282] In certain embodiments of the invention, the compound is a
small molecule (less than 10 kDa), e.g., a non-peptide small
molecule.
6. EXAMPLES
6.1. Example I
Kinase Activity Assay on Microarray Materials & Reagents
[0283]
3 Materials/Equipment/Reagents Vendor Part Number
Disposables/Reagents Gamma-AT.sup.33P (10 .mu.Ci/.mu.l, Perkin
Elmer NEG602H250UC 250 .mu.Ci) Histone, Calf Thymus Calbiochem
38205 Casein Sigma C-4032 Myelin Basic Protein Sigma M-1891
Poly-glutamic acid-tyrosine Sigma P-2075 PBS Tablets American
AB11108 Bioanalytical Tween-20 American AB02038 Bioanalytical 60
.times. 24 mm Hybridization Cover Schleicher & 10 484 907 slips
Schuell Equipment Cyclone Phospho-imager Perkin Elmer B431220 8
.times. 10 Autoradiography Cassettes Fisher FB-XC-810 Phosphor
Storage Screens (MS) Perkin Elmer 7001723 Lab Rotator Lab-Line 1314
Instruments Eppendorf Centrifuge (5810) Fisher Scientific
05-400-60
[0284] Reagent/Stock Preparation
[0285] Kinase Substrate Stocks
[0286] Dissolve protein substrates in 20 mM Tris to a final
concentration of 10 mg/mL.
[0287] 1 L of 1.times.PBS
[0288] Dissolve 5 PBS tablets in 1 L dH2O.
[0289] Mix thoroughly.
[0290] 1 L PBST
[0291] Dissolve 5 PBS tablets in IL dH2O.
[0292] Add 1 mL Tween-20.
[0293] Mix thoroughly.
[0294] Kinase Assay Dilution Buffer
[0295] 20 mM MOPS, pH 7.2
[0296] 25 mM b-glycerol phosphate
[0297] 5 mM EGTA
[0298] 1 mM sodium orthovanadate
4 Assay Solution (1 ml nominal - total = .about.1.1 ml) In 1 ml of
Kinase Assay Dilution Buffer, add (.about.final concentration) 1
.mu.l of 1 M DTT (1 mM) 1 .mu.l of 30% BSA (3 mg/ml) 1 .mu.l of 1 M
MnCl.sub.2 (1 mM) 1 .mu.l of 1 M CaCl.sub.2 (1 mM) 25 .mu.l of 1 M
MgCl.sub.2 (25 mM)
[0299] Methods
[0300] Step 1: Coating of Slides with Kinase Substrates
[0301] To coat slides with kinase substrate, the substrates are
diluted to 10 ng/.mu.L in 1.times.PBS and 180-200 .mu.L of
substrate solution are pipetted onto one slide, e.g., a glass
slide, aldehyde treated slides (TeleChem International, Inc.),
nitrocellulose-coated slides (Schleicher & Schuell), slides
with an amino-silane surface (Corning). A second slide is then
placed on top of the first slide so that the sides to be printed
with kinases face each other. Care should be taken that the liquid
covers the entire slide and that there are no air bubbles. The
slides are placed in a 50 mL conical tube, making sure they are
laying flat and incubated at 4.degree. C. for one hour to several
days.
[0302] Alternatively, substrates may be printed on the slides using
a microarrayer, wherein the samples are kept at 4.degree. C. The
substrates should be diluted in the proper printing buffer. The
spot size should be 150-200 .mu.m, and the spacing should be
between 0.5 and 1 mm. After printing, incubate at 4.degree. C. for
one hour to several days.
[0303] Step 2: Washing and Blocking of Coated Slides
[0304] The substrate-coated or substrate-printed slides obtained in
step 1 are removed from the conical tubes and placed in a slide
staining dish. Subsequently, approximately 100 mL of PBST are added
to the dish. The slides are then washed for one hour at 4.degree.
C. with shaking. The PBST is then discarded and the slides are
gently rinsed with dH2O using a squirt bottle. After rinsing, the
slides are placed into a slide boxes and centrifuged at 4000 rpm
for one minute. The slides are then stored at 4.degree. C. until
printing with kinase.
[0305] Step 3: Printing of Kinases on Substrate-Coated Slides
[0306] Kinases are diluted in the proper printing buffer. The
concentration should be between 1 and 10 ng/.mu.L. The kinases are
printed on the substrate-coated slides obtained in step 1 and 2
using a microarrayer. The spot size should be 150-200 .mu.m, and
the spacing should be between 0.5 and 1 mm. If the substrate is
printed on the slides, the spacing of the kinase array should match
that of the substrate array (i.e., the kinases should be printed on
top of the substrate). The slides can be stored at 4.degree. C.
until the kinase activity assay is performed.
[0307] Step 4: Assay of Kinase Activity on Microarray
[0308] 1 mL of kinase assay buffer for every 12 glass slides to be
probed is prepared. 6 .mu.L of gamma-AT.sup.33P (10 .mu.Ci/.mu.L)
are added to the assay buffer. The slides are placed in 50 mL
conical tubes, laying flat, proteins facing up. 70 .mu.L of the
kinase assay buffer with gamma-AT.sup.33P are added onto each
slide. Using tweezers, the slide is covered with a hybridization
slip, making sure that the solution completely covers the
microarray. The conical tube is then closed and placed in a
30.degree. C. incubator. Care should be taken that the slide is
laying flat. The reaction is then incubated for 90 minutes.
Subsequently, the tubes are removed from the incubator.
Approximately 40 mL of dH.sub.2O are added to each tube and, using
the tweezers, the hybridization slip is removed, the tube is closed
and inverted several times for 1-2 minutes to rinse the slide
inside the conical tube. The wash solution is then discarded.
Approximately 40 mL of dH.sub.2O are added again to each tube, the
tubes are closed and inverted several times for 1-2 minutes, the
wash solution is discarded. The slides are then removed from the
tubes and place in a slide box and centrifuged at 4000 rpm for 1-2
minutes.
[0309] Phosphor screen (suitable for .sup.33P) is re-activated for
each membrane by exposing it to light for at least 30 minutes. A
piece of filter paper is placed in an autoradiography cassette and
the dried slides are placed on the filter paper, facing up. The
slides are covered with a piece of clear plastic film (such as
SaranWrap). The phosphor screen is placed on top of the SaranWrap,
facing the slides. The cassette is then closed and locked and
exposed for a few hours to a couple of days, depending on the
activity. In a dark room (or a room with dim light), the cassette
is opened and the phosphor screen is removed. The phosphor screen
is then mounted on the Cyclone rotor and scanned at 600 dpi.
[0310] An exemplary autoradiograph of a kinase reaction of the
present invention is shown in FIG. 1. The data shown in FIG. 1 were
obtained essentially using the method described above. As shown in
FIG. 1, the presence of substrate is required for the kinase
reaction. Thus, the signal obtained in this experiment is due to
specific phosphorylation of the substrate and not due to
autophosphorylation or binding of the labeled ATP to some of the
enzymes.
[0311] The data shown in FIG. 2 demonstrate that treatment of the
slide with aldehyde improves the signal-to-noise ratio. The
experiments were conducted essentially using the method described
above but with different types of slides. The aldehyde-treated
slides were obtained from TeleChem International, Inc. The slide
shown as FAST is a nitrocellulose coated slide and was obtained
from Schleicher & Schuell. The slide shown as GAPS is coated
with an amino-silane surface and was obtained from
Corning.RTM..
[0312] Safety Considerations
[0313] 1. The operator must follow proper procedures and use
cautions when handling radioactive materials.
[0314] 2. Before using the microarrayer, the operator should be
trained to avoid injuries to the person and/or damages to the
machine.
6.2. Example II
Inhibitor Specificity Profiling
[0315] Fifty different kinases were immobilized on a slide together
with a substrate as described in section 6.1. A mixture of Myelin
Basic Protein (MBP), histone and casein was used as substrate. The
kinase reactions were performed in the presence of H89 inhibitor,
Rottlerin inhibitor or PP2 inhibitor (FIG. 3). The inhibitors were
obtained from Calbiochem. The PP2 inhibitor is an inhibitor of
tyrosine kinases. The concentration of inhibitor was 100 .mu.m for
each inhibitor. The control reaction was performed in the absence
of inhibitor. The specificity of the assay is demonstrated by the
fact that PP2 inhibitor strongly inhibits tyrosine kinases (see
circled data points in FIG. 3).
6.3. Example III
Dose-Response Analyses
[0316] Microarrays were prepared with 10 wells/slide, wherein the
kinases EPHB3, FYN, and PRKCD and their substrate were immobilized
in each well. The slide was coated with substrate essentially as
described in section 6.1. Subsequently, a gasket with 10 openings
was applied to the surface of the slide thereby creating 10 wells,
i.e., the gasket provides the barriers between the wells. The
accession numbers for the different kinases in the NCBI database
are: for FYN: NM.sub.--002037; for PRKCD: NM.sub.--006254; and for
EPHB3: NM.sub.--004443. A mixture of Myelin Basic Protein (MBP),
histone and casein was used as substrate. The kinase reaction was
performed in each well with a different concentration of PP2
inhibitor. The dose-response curve is shown in FIG. 4a. The data
show that PP2 strongly inhibits the tyrosine kinases FYN and EPHB3
but not the serine/threonine kinase PRKCD. In a second exeriment,
the kinase reaction was performed in each well with a different
concentration of Staurosporin. The dose-response curve shown in
FIG. 4b demonstrates that Staurosporine strongly inhibits PRKCD and
FYN but not EPHB3.
6.4 Example IV
Comprehensive Inhibitor Assays
[0317] The surface of a slide is coated with substrate within the
wells of a multiwell array. The surface is coated with substrate,
and washed and blocked as described in section 6.1. Subsequently, a
gasket with openings is applied to the surface of the slide thereby
creating wells, i.e., the gasket provides the barriers between the
wells.
[0318] The kinases are printed on the surface by the following
procedure. The dimensions of the wells of the multi-well array used
are obtained and the areas on the slides that will match the wells
are defined. These numbers are used to calibrate the microarrayer
so that the printed spots will locate within the wells. The wells
are formed later by placing the gasket with openings on top of the
surface of the solid support.
[0319] The number of proteins that can be printed per well depends
on the dimension of the well and the spacing required. The chambers
made by Scleicher & Schuell and Grace Bio-labs have 7000
.mu.m.times.7000 .mu.m wells and allow up to 12.times.12 spots/well
printed if the spacing is 500 .mu.m. At least 4 replicate per
kinase is recommended for quantitative experiments.
[0320] The plate of kinases to be printed is made so that the
printing pins pick up the identical kinase preparation (identical
volume, concentration, buffer components, etc.) at the same time.
This will ensure comparable results among the arrays. In addition,
kinase activities should be assessed and normalized to give uniform
signals within the array. The kinases are printed onto the slide as
described in section 6.1.
[0321] The kinase assay is performed by removing the plastic
covering from sticky side of the chamber, placing the chamber
carefully on the printed slides, aligning the wells to the printed
areas. The chamber is placed on the slide to make a tight seal
between wells. Subsequently, the kinase assay buffer with
gamma-AT.sup.33P is prepared as described in section 6.1.
Inhibitors (or other molecules of interest or concentrations of the
same molecule) are prepared in aliquots. The cover slip is removed
from the chamber, thereby exposing the wells. Appropriate amounts
of inhibitor and kinase assay buffer is added to wells (volumes
that will cover the well but not exceed the well capacity). The
cover slip is placed on the slide and the entire slide/chamber
assembly is placed in a 50 ml tube. The slides are incubated at
30.degree. C. for 90 minutes, making sure the slides sit flat. The
slides are washed as described in section 6.1. The chamber is
removed from the tube using a pair of tweezers and the wash
procedure is repeated once. The kinase reaction is evaluated as
described in section 6.1.
[0322] The data shown in FIG. 5 were obtained by the procedure
described in this section. The type of inhibitor used in the
reactions is shown on the side of the slide shown in FIG. 5. A
reaction was performed on the same slide without an inhibitor as
control (lower right part of the slide).
[0323] References Cited
[0324] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication or patent or patent application
was specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
[0325] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled.
* * * * *