Protein expression yield enhancement in cell-free protein synthesis systems by addition of antifoam agents

• US 8,298,759 B2
• Filed: 03/21/2005
• Issued: 10/30/2012
• Est. Priority Date: 03/25/2004
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Voloshin, Alexei M., Swartz, James Robert
• Primary Examiner(s): Kim, Young J
• Application Number:
  US10/599,310
• Publication Number:
  US 20090042244A1
• Time in Patent Office: 2,780 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00

Abstract

Compositions and methods are provided for the in vitro synthesis of biological molecules in reaction mixtures comprising anti-foam agents. The reaction mix comprising antifoam agent may be a scaled up reaction, e.g. in reaction volume greater than at least about 15 μl. Reactions may be performed in various reactors, as known in the art, which include stirred reactors, bubble-column reactors; and the like.

Claims

1. A method for translation of mRNA to produce polypeptides, the method comprising:

synthesizing said polypeptides in a cell-free reaction mixture of greater than about 15 μ

l volume, comprising an antifoam agent at a concentration of at least 0.00007%, and not more than 0.007% by weight, wherein the antifoam agent is other than a detergent.

2. The method of claim 1 wherein said synthesizing also comprises transcription of mRNA from a DNA template.

3. The method of claim 1, wherein said reaction mix comprises a volume of greater than 100 μ

l.

4. The method of claim 3, wherein said reaction has a yield that is at least about 90% of the yield in a comparable reaction of less than 15 μ

l volume.

5. The method of claim 1 wherein oxidative phosphorylation is activated in the cell-free reaction mixture.

6. The method of claim 1 wherein said reaction mixture comprises a volume of greater than 1000 μ

l.

7. The method of claim 1, wherein said synthesizing is performed in a reactor.

8. The method of claim 7, wherein the reactor is a bubble reactor.

9. The method of claim 1, wherein the antifoam agent is selected from alkyl polyoxyalkylene glycol ethers;

siloxane polymers; and

mixtures of organic non-silicone polypropylene based polyether dispersions.

10. A method for translation of mRNA to produce polypeptides, the method comprising:

synthesizing said polypeptides in a cell free reaction mixture of greater than about 15 μ

l volume, comprising;

a cell extract;

a template for production of the mRNA and/or polypeptides;

monomers for the mRNA and/or polypeptides to be synthesized; and

co-factors, enzymes and reagents that are necessary for the synthesis; and

an anti-foam agent at a concentration of at least 0.00007%, and not more than 0.007% by weight, wherein the antifoam agent is other than a detergent.

11. The method of claim 10, wherein the antifoam agent is selected from alkyl polyoxyalkylene glycol ethers;

siloxane polymers; and

mixtures of organic non-silicone polypropylene based polyether dispersions.

12. A reaction mixture for cell-free translation of mRNA to produce polypeptides, comprising:

a cell extract;

mRNA;

monomers for the polypeptide to be synthesized; and

co-factors, enzymes and reagents that are necessary for the synthesis; and

an anti-foam agent other than a detergent at a concentration of at least 0.00007%, and not more than 0.007% by weight.

13. The reaction mixture of claim 12, wherein the antifoam agent is selected from alkyl polyoxyalkylene glycol ethers;

siloxane polymers; and

mixtures of organic non-silicone polypropylene based polyether dispersions.