Expression of biologically active proteins in a bacterial cell-free synthesis system using bacterial cells transformed to exhibit elevated levels of chaperone expression

A bacterial cell free synthesis system for expressing biologically active proteins comprising:

• i) a cell free S30 extract of E. coli bacteria having an active oxidative phosphorylation system, containing biologically functioning tRNA, amino acids and ribosomes necessary for cell free protein synthesis and wherein an exogenous DsbC disulfide isomerase and an exogenous FkpA prolyl isomerase were expressed in the bacteria at a level of at least 1 g/liter of extract;
• and
• ii) a nucleic acid encoding a protein of interest, wherein the protein of interest comprises a disulfide bond and a proline residue,
• where said bacterial cell free synthesis system expresses the protein of interest to a concentration of at least about 100 mg/L.

A method of expressing properly folded, biologically active proteins in a bacterial cell free synthesis system comprising the steps of:

• i)
• combining a bacterial extract with a nucleic acid encoding a protein of interest comprising a disulfide bond and a proline residue;
• and
• ii) incubating the bacterial extract with the nucleic acid under conditions permitting the expression and proper folding of the protein of interest,
• wherein the bacterial extract is an S30 extract of E. coli bacteria comprising biologically functioning tRNA, amino acids, ribosomes necessary for cell free protein synthesis, a DsbC protein disulfide isomerase and a FkpA peptidyl-prolyl cis-trans isomerase, wherein the DsbC protein disulfide isomerase and the FkpA peptidyl-prolyl cis-trans isomerase are present at a concentration of at least about 1 g/liter of extract.

A bacterial cell free synthesis system for expressing biologically active proteins in a comprising:

• i) a cell free S30 extract of E. coli bacteria having an active oxidative phosphorylation system, containing biologically functioning tRNA, amino acids and ribosomes necessary for cell free protein synthesis, and further including a DsbC protein disulfide isomerase and a FkpA peptidyl-prolyl cis-trans isomerase,
• wherein the protein disulfide isomerase and the peptidyl-prolyl cis/trans isomerase are present at a concentration of at least 1 g/liter of extract;
• and
• ii) a nucleic acid encoding a protein of interest comprising a disulfide bond and a proline residue,
• wherein said bacterial cell free synthesis system expresses a protein of interest to a concentration of at least about 100 mg/L.