Methods of in vitro protein synthesis

Patent metadata

Field	Value
Publication number	US-9410170-B2
Application number	US-57971104-A
Country	US
Kind code	B2
Filing date	Nov 18, 2004
Priority date	Nov 20, 2003
Publication date	Aug 9, 2016
Grant date	Aug 9, 2016

Abstract

Improved methods are provided in vitro synthesis of biological molecules, providing for improved yields, lowered costs, and enhanced utility. Improved yield and lowered cost is obtained by the use of a phosphate free energy source in the presence of exogenous phosphate, and optionally in the absence of exogenous nucleoside triphosphates.

First claim

What is claimed is:

1. A method for synthesis of polynucleotides and/or polypeptides in a cell-free reaction mixture initially comprising a bacterial cell extract; a template for production of the polynucleotides and/or polypeptide; monomers for the polynucleotides and/or polypeptide to be synthesized, and such co-factors, enzymes and other reagents that are necessary for the synthesis; the method comprising:
synthesizing said polynucleotides and/or polypeptides in a cell-free reaction mixture modified to include:
at least 10 mM of a phosphate-free energy source wherein said phosphate free energy source is glucose or glutamate; nucleoside monophosphates in the absence of exogenous nucleoside triphosphates; and exogenous inorganic phosphate salts at a concentration of at least about 1 mM.

2. The method of claim 1 , wherein the exogenous inorganic phosphate is provided at a concentration of from about 1 mM to about 20 mM.

3. The method of claim 1 , wherein said phosphate is provided as potassium phosphate, magnesium phosphate, or ammonium phosphate.

4. The method of claim 1 , wherein said synthesis comprises translation of mRNA to produce polypeptides.

5. The method of claim 4 , wherein said synthesis also comprises transcription of mRNA from a DNA template.

6. The method of claim 1 , wherein said synthesis of polynucleotides and/or polypeptides is performed as a batch reaction.

7. The method of claim 1 , wherein said synthesis of polynucleotides and/or polypeptides is performed as a continuous reaction.

8. The method of claim 1 , wherein said reaction mix comprises an extract from E. coli grown in glucose containing medium.

9. The method of claim 8 , wherein said E. coli are grown in glucose and phosphate containing medium.

10. The method of claim 1 , wherein said reaction mix comprises magnesium at a concentration of from about 5 mM to about 20 mM.

11. The method of claim 1 , wherein said reaction mix is substantially free of polyethylene glycol.

12. The method according to claim 11 , wherein said reaction mix comprises one or more of spermine, spermidine and putrescine.

13. The method of claim 1 , wherein the reaction mixture yields over 400 μg/mL of the synthesized polypeptide.

Assignees

• Calhoun Kara
• Jewett Michael Christopher
• Swartz James Robert
• Univ Leland Stanford Junior

Inventors

• Calhoun Kara
• Jewett Michael Christopher
• Swartz James Robert

Classifications

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C07K1/00 Primary

  General methods for the preparation of peptides {, i.e. processes for the organic chemical preparation of peptides or proteins of any length} · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title