High throughput protein production screening

• US 20050019807A1
• Filed: 07/01/2004
• Published: 01/27/2005
• Est. Priority Date: 07/24/2003
• Status: Active Grant

• Current Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Coleman, Matthew A., Segelke, Brent W., Beernink, Peter T.
• Granted Patent: US 7,585,815 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins

Abstract

Methods, compositions, and kits for the cell-free production and analysis of proteins are provided. The invention allows for the production of proteins from prokaryotic sequences or eukaryotic sequences, including human cDNAs using PCR and IVT methods and detecting the proteins through fluorescence or immunoblot techniques. This invention can be used to identify optimized PCR and WT conditions, codon usages and mutations. The methods are readily automated and can be used for high throughput analysis of protein expression levels, interactions, and functional states.

Claims

1. A method for parallel analysis of a plurality of protein expression levels, said method comprising:

carrying out in a plurality of reaction mixtures in vitro translation reactions in the presence of an aminoacyl tRNA, said amino acyl tRNA comprising a label on the amino acid moiety of said amino acyl tRNA to generate a plurality of labeled reaction products;

spotting said plurality of labeled reaction products onto a substrate; and

detecting said plurality of labeled reaction products.

2. The method of claim 1, wherein said label is a fluorescent label.

3. The method of claim 1, wherein said reaction mixtures are contained within wells of a multiwell plate.

4. The method of claim 1, further comprising carrying out an in vitro transcription reaction in said reaction mixtures.

5. The method of claims 1 and 4, wherein said reaction mixtures are spotted onto said substrate without an intervening purification step.

6. The method of claim 1, wherein said substrate is a membrane.

7. The method of claim 6, wherein said membrane comprises PVDF

8. The method of claim 1, wherein said substrate is glass.

9. The method of claim 8, wherein said glass substrate is coated.

10. The method of claim 9, wherein said coating comprises poly-L-lysine.

11. The methods of claim 1, wherein said detecting step comprises use of an antibody.

12. The method of claim 11, wherein said antibody includes an enhanced chemiluminescent label.

13. A method of parallel analysis of protein interactions, comprising the steps of:

carrying out in a plurality of reaction mixtures in vitro translation reactions;

spotting said reaction mixtures onto a substrate;

exposing said substrate to a test protein; and

determining whether said test protein binds to an in vitro translated reaction product present in said reaction mixtures.

14. The method of claim 13, further comprising carrying said plurality of in vitro translation reactions in the presence of an aminoacyl tRNA, said amino acyl tRNA comprising a label on the amino acid moiety of said amino acyl tRNA to generate a plurality of labeled reaction products.

15. The method of claim 14, wherein said label is a fluorescent label.

16. The method of claim 13, wherein said reaction mixtures are contained within wells of a multiwell plate.

17. The method of claim 13, further comprising carrying out an in vitro transcription reaction in said reaction mixtures.

18. The method of claims 13 and 17, wherein said reaction mixtures are spotted onto said substrate without an intervening purification step.

19. The method of claim 13, wherein said substrate is a membrane.

20. The method of claim 19, wherein said membrane comprises PVDF.

21. The method of claim 19, wherein said membrane comprises nylon.

22. The method of claim 19, wherein the membrane comprises nitrocellulose.

23. The method of claim 13, wherein said substrate is glass.

24. The method of claim 23, wherein said glass substrate is coated.

25. The method of claim 24, wherein said coating comprises poly-L-lysine.

26. The method of claim 24, wherein said coating comprises gamma aminopropyl silane.

27. The method of claim 13, wherein said determining step comprises exposing said substrate to an antibody directed against said test protein.

28. The method of claim 27, wherein said antibody includes an enhanced chemiluminescent label.

29. A kit, comprising, reagents for carrying out an in vitro translation reaction, a reagent for labeling an in vitro translation reaction product, at least one of a wash buffer or a listing of a wash buffer composition, and instructions for detecting said labeled in vitro translation reaction by a method comprising spotting said labeled reaction product onto a substrate and washing said substrate with said wash buffer.