Methods of in vitro protein synthesis
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.
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.