Protein arrays and methods of use thereof
The present invention provides human protein arrays that include at least 1000 human proteins. In another embodiment, the present invention provides a method for identifying a substrate of an enzyme, comprising contacting the enzyme with a positionally addressable array comprising at least 100 proteins immobilized on functionalized glass surface, and identifying a protein on the positionally addressable array that is bound and/or modified by the enzyme, wherein a binding or modifying of the protein by the enzyme indicates that the protein is a substrate for the enzyme. In additional embodiments, provided herein are methods for making an array of at least 1000 human proteins under non-denaturing conditions, including human proteins that are difficult to express and/or difficult to isolate in a non-denatured state.
1 . A positionally addressable array comprising 100 human proteins from the proteins listed in Table 9, Table 11, and Table 13, immobilized on a substrate.
2 . The positionally addressable array of claim 1 , wherein the array comprises 500 human proteins from the proteins listed in Table 9, Table 11, and Table 13.
3 . The positionally addressable array of claim 1 , wherein the array comprises 1000 human proteins from the proteins listed in Table 9, Table 11, and Table 13.
4 . The positionally addressable array of claim 1 , wherein the array comprises 2500 human proteins from the proteins listed in Table 9, Table 11, and Table 13.
5 . The positionally addressable array of claim 1 , wherein the array comprises 5000 human proteins from the proteins listed in Table 9, Table 11, and Table 13.
6 . The positionally addressable array of claim 1 , wherein the array comprises 100 of the membrane proteins of Table 15.
7 . A positionally addressable array of claim 1 , wherein the array comprises 250 of the membrane proteins of Table 15.
8 . The positionally addressable array of claim 7 , wherein the array comprises 50 of the transmembrane proteins of Table 16.
9 . The positionally addressable array of claim 7 , wherein the array comprises all of the transmembrane proteins of Table 16.
10 . The positionally addressable array of claim 7 , wherein the array comprises at least 25 of the G protein coupled receptors (GPCRs) of Table 17.
11 . The positionally addressable array of claim 10 , wherein the array comprises all of the GPCRs of Table 17.
12 . The positionally addressable array of claim 1 , wherein proteins are present on the array at a density of between 500 proteins/cm 2 and 10,000 proteins/cm 2 .
13 . The positionally addressable array of claim 1 , wherein the proteins are non-denatured proteins.
14 . The positionally addressable array of claim 1 , wherein the proteins are full-length proteins.
15 . The positionally addressable array of claim 1 , wherein the proteins are non-denatured, full-length, recombinant fusion proteins comprising a tag.
16 . The positionally addressable array of claim 1 , wherein the substrate is a functionalized glass slide.
17 . The positionally addressable array of claim 16 , wherein the functionalized glass slide comprises a polymer comprising an acrylate group, wherein the polymer overlays a glass surface.
18 . The positionally addressable array of claim 17 , wherein the substrate is a Protein slides II functionalized glass protein microarray substrate available from Full Moon Biosystems
19 - 22 . (canceled)
23 . A method for identifying a substrate of an enzyme, comprising contacting the enzyme with a positionally addressable array comprising at least 100 proteins immobilized on a functionalized glass slide, and identifying a protein on the positionally addressable array that is modified by the enzyme, wherein a modifying of the protein by the enzyme indicates that the protein is a substrate for the enzyme.
24 . The method of claim 23 , wherein the functionalized glass slide comprises a three-dimensional porous surface comprising a polymer overlaying a glass surface.
25 . The method of claim 24 , wherein the three-dimensional porous surface comprises a polymer comprising acrylate, overlaying a glass surface.
26 . The method of claim 25 , wherein the functionalized glass substrate comprises multiple functional protein-specific binding sites.
27 . The method of claim 26 , wherein the substrate is a Protein slides II protein microarray substrate available from Full Moon Biosystems, Inc.
28 . The method of claim 23 , wherein the enzyme activity is a chemical group transferring enzymatic activity.
29 . The method of claim 23 , wherein the enzyme activity is kinase activity, protease activity, phosphatase activity, glycosidase, or acetylase activity.
30 . The method of claim 23 , wherein the enzyme activity is kinase activity.
31 - 43 . (canceled)
44 . A method for making an array of proteins, comprising:
cloning each open reading frame from a population of open reading frames into a baculovirus vector to generate a recombinant baculovirus vector comprising a promoter that directs expression of a fusion protein comprising the open reading frame linked to a tag;
expressing the fusion proteins generated for each of the population of open reading frames using insect cells;
isolating the fusion proteins using affinity chromatography directed to the tag; and
spotting the isolated proteins on a substrate.
45 . The method of claim 44 , wherein the cells are sf9 cells.
46 . The method of claim 44 , wherein the array of proteins comprises 1000 full length mammalian proteins.
47 . The method of claim 46 , wherein the proteins are human proteins.
48 . The method of claim 47 , wherein the proteins comprise at least 250 membrane proteins of Table 15.
48 . The method of claim 48 , wherein the proteins comprise at least 50 transmembrane proteins of Table 16.
50 . The method of claim 49 , wherein the proteins comprise at least 25 G-protein coupled receptor proteins of Table 17.
51 . The method of claim 44 , wherein the tag is a GST tag.
52 . The method of claim 48 , wherein the proteins are expressed, isolated, and spotted in a high-thoughput manner, and under non-denaturing conditions.
53 - 61 . (canceled)