Methods of in vitro protein synthesis
View Patent ↗Biological macromolecules are synthesized in vitro under conditions and in a reaction composition wherein oxidative phosphorylation is activated and protein folding is improved.
1. A method for in vitro transcription of mRNA and/or translation of polypeptides, the method comprising:
synthesizing said mRNA and/or polypeptides in a transcription and/or translation reaction mix substantially free of polyethylene glycol, comprising:
an extract from E. coli cells comprising membrane vesicles containing respiratory chain components; components of polypeptide and/or mRNA synthesis machinery; a template for transcription of said mRNA and/or translation of said polypeptide; monomers for synthesis of said mRNA and/or polypeptides; and co-factors, enzymes and other reagents necessary for said transcription and/or translation;
magnesium at a concentration of from about 5 mM to about 20 mM;
wherein oxidative phosphorylation, which is sensitive to electron transport chain inhibitors, is activated in said reaction mix.
2. The method of claim 1 , wherein transcription of mRNA and/or translation of polypeptides is at least two fold higher than synthesis in the absence of said oxidative phosphorylation.
3. The method according to claim 1 , wherein transcription of mRNA and/or translation of polypeptides is at least three fold higher than synthesis in the absence of said oxidative phosphorylation.
4. The method of claim 1 wherein said transcription of mRNA and/or translation of polypeptides is performed as a batch reaction.
5. The method of claim 1 , wherein said transcription of mRNA and/or translation of polypeptides is performed as a continuous reaction.
6. A method for in vitro transcription of mRNA and/or translation of polypeptides, the method comprising:
synthesizing said mRNA and/or polypeptides in a transcription and/or translation reaction mix substantially free of polyethylene glycol, comprising:
an extract from E. coli cells grown in glucose and phosphate containing medium comprising components of polypeptide and/or mRNA synthesis machinery; a template for transcription of said mRNA and/or translation of said polypeptide; monomers for synthesis of said mRNA and/or polypeptides; and co-factors, enzymes and other reagents necessary for said transcription and/or translation;
magnesium at a concentration of from about 5 mM to about 20 mM;
at least one of spermine or spermidine at a concentration of at least about 1 mM;
wherein oxidative phosphorylation, which is sensitive to electron transport chain inhibitors, is activated in said reaction mix.
7. The method of claim 6 wherein said synthesis is performed as a batch reaction.
8. The method of claim 6 , wherein said synthesis of polypeptides is performed as a continuous reaction.
9. The method of claim 6 , wherein transcription of mRNA and/or translation of polypeptides is at least two fold higher than synthesis in the absence of said oxidative phosphorylation.
10. The method according to claim 6 , wherein transcription of mRNA and/or translation of polypeptides is at least three fold higher than synthesis in the absence of said oxidative phosphorylation.