Enhanced in vitro synthesis of active proteins containing disulfide bonds
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsJames Robert Swartz, Dong-Myung Kim
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Abstract
Compositions and methods are provided for the enhanced in vitro synthesis of polypeptides containing disulfide bonds. In order to improve the performance of in vitro protein synthesis reactions, pre-treatment and redox buffering of the reaction mix is performed in order to optimize the redox potential. Exogenous enzymes that enhance protein folding and disulfide bond formation may also be added to the reaction.
Claims12
A method for in vitro synthesis of polypeptides comprising at least one disulfide bond, in a reaction mix comprising a biological extract comprising components of polypeptide synthesis machinery, wherein such components are capable of expressing a nucleic acid encoding a desired polypeptide, the improvement comprising:
- synthesizing said polypeptide in a reaction mix wherein said biological extract has been pre-treated with a sulfhydryl inactivating agent that alkylates or acetylated free sulfhydryl groups;
- isolating said polypeptide from said reaction mixture, wherein the amount of said polypeptide that is properly folded is enhanced relative to polypeptide synthesized in the absence of pretreatment with said sulfhydryl inactivating agent.
A method for in vitro synthesis of polypeptides comprising at least one disulfide bond in a reaction mix comprising a biological extract comprising components of polypeptide synthesis machinery, wherein such components are capable of expressing a nucleic acid encoding a desired polypeptide, the improvement comprising:
- synthesizing said polypeptide in a reaction mix wherein said biological extract has been pre-treated with a sulfhydryl inactivating agent that alkylates or acetylated free sulfhydryl groups, wherein said reaction mix further comprises a redox buffer;
- isolating said polypeptide from said reaction mixture, wherein the amount of said polypeptide that is properly folded is enhanced relative to polypeptide synthesized in the absence of pretreatment with said sulfhydryl inactivating agent.
A method for in vitro synthesis of polypeptides comprising at least one disulfide bond in a reaction mix comprising a biological extract comprising components of polypeptide synthesis machinery, wherein such components are capable of expressing a nucleic acid encoding a desired polypeptide, wherein such components are capable of expressing a nucleic acid encoding a desired polypeptide, the improvement comprising:
- synthesizing said polypeptide in a reaction mix comprising a redox buffer, wherein said biological extract has been pre-treated with a sulfhydryl inactivating agent that alkylates or acetylated free sulfhydryl groups, wherein said reaction mixture comprises a foldase enzyme;
- isolating said polypeptide from said reaction mixture, wherein the amount of said polypeptide that is properly folded is enhanced relative to polypeptide synthesized in the absence of pretreatment with said sulfhydryl inactivating agent.
Dependencies are inferred from the claim text.