High throughput protein production screening

• US 7,585,815 B2
• Filed: 07/01/2004
• Issued: 09/08/2009
• Est. Priority Date: 07/24/2003
• Status: Expired due to Fees

• Current Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Original Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Inventors: Beernink, Peter T., Coleman, Matthew A., Segelke, Brent W.
• Primary Examiner(s): Steele; Amber D.
• Application Number:
  US10/884,783
• Publication Number:
  US 20050019807A1
• Time in Patent Office: 1,895 Days
• Field of Search: 506/3, 506/4, 435/6, 435/68.1, 530/412
• US Class Current: 506/3
• 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 in vitro transcription/translation reactions, said method comprising:

carrying out said plurality of in vitro transcription/translation reactions in a plurality of reaction mixtures in wells of a multiwell plate to generate a plurality of reaction products, wherein each non-control reaction mixture comprises cell-free protein expression reagents and at least one nucleic acid template;

spotting said plurality of reaction mixtures including reaction products onto a substrate wherein the substrate is optionally coated, wherein said reaction products bind directly to said substrate or said coated substrate, wherein said substrate comprises a membrane or a glass slide and wherein said spotting is carried out without use of a separation step that provides information about molecular weights of reaction products and without an intervening purification step; and

detecting said plurality of in vitro transcription/translation reaction products bound to said substrate.

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

3. The method of claim 2, wherein said membrane comprises PVDF.

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

5. The method of claim 1, wherein said coated substrate is a coated glass slide.

6. The method of claim 5, wherein said coating comprises poly-L-lysine.

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

8. The method of claim 7, wherein said antibody includes an enhanced chemiluminescent label.

9. The method of claim 1, further comprising carrying out said plurality of in vitro transcription/translation reactions in the presence of a labeled aminoacyl tRNA, said labeled amino acyl tRNA comprising a label on the amino acid moiety of said amino acyl tRNA to generate said plurality of in vitro transcription/translation reaction products that are labeled.

10. The method of claim 9, wherein said label is a fluorescent label.

11. The method of claim 1, wherein said plurality of in vitro transcription/translation reactions comprises at least 13 reactions.

12. The method of claim 1, wherein said plurality of in vitro transcription/translation reactions comprises at least 48 reactions.

13. The method of claim 1, wherein said plurality of in vitro transcription/translation reactions comprises at least 96 reactions.

14. The method of claim 1, wherein said plurality of in vitro transcription/translation reactions comprises at least 224 reactions.

15. The method of claim 1, wherein said plurality of in vitro transcription/translation reactions comprises at least 384 reactions.

16. The method of claim 1, wherein said cell-free expression reagents comprise an E. coli lysate and said nucleic acid template comprises DNA.

17. The method of claim 1, further comprising amplifying a plurality of nucleic acid samples to generate the nucleic acid templates prior to the step of carrying out a plurality of in vitro transcription/translation reactions.