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

Abstract

The present disclosure describes methods and systems for improving the expression of a properly folded, biologically active protein of interest in a cell free synthesis system. The methods and systems use a bacterial cell free extract having an active oxidative phosphorylation system, and include an exogenous protein chaperone. The exogenous protein chaperone can be expressed by the bacteria used to prepare the cell free extract. The exogenous protein chaperone can be a protein disulfide isomerase and/or a peptidyl-prolyl cis-trans isomerase. The inventors discovered that the combination of a protein disulfide isomerase and a peptidyl-prolyl cis-trans isomerase produces a synergistic increase in the amount of properly folded, biologically active protein of interest.

1 .- 37 . (canceled) 
     
     
         38 . A bacterial strain comprising genomic integration of one or more expression cassettes that express high levels of an exogenous disulfide isomerase or an exogenous prolyl isomerase. 
     
     
         39 . The bacterial strain of  claim 38 , wherein the one or more expression cassettes comprise a gene encoding the exogenous disulfide isomerase or the exogenous prolyl isomerase operably linked to a promoter. 
     
     
         40 . The bacterial strain of  claim 38 , wherein the exogenous disulfide isomerase is DsbC and the exogenous prolyl isomerase is FkpA. 
     
     
         41 . The bacterial strain of  claim 40 , wherein the stain comprises two copies of the dsbC gene integrated into the chromosome. 
     
     
         42 . The bacterial strain of  claim 41 , wherein the stain further comprises a plasmid comprising two copies of the FkpA gene operably linked to a promoter. 
     
     
         43 . The bacterial strain of  claim 40 , wherein the stain comprises two copies of the FkpA gene integrated into the chromosome. 
     
     
         44 . The bacterial strain of  claim 40 , wherein the stain comprises two copies of the dsbC gene and two copies of the FkpA gene integrated into the chromosome. 
     
     
         45 . The bacterial strain of  claim 38 , wherein the strain is an  E. coli  bacterial strain. 
     
     
         46 . The bacterial strain of  claim 38 , wherein the strain comprises an ompT1 sensitive RF1 protein. 
     
     
         47 . A cell free extract prepared from the bacterial strain of  claim 38 . 
     
     
         48 . A method for producing an exogenous protein chaperone, comprising culturing the bacterial strain of  claim 38  under conditions that permit the overexpression of the exogenous protein chaperones. 
     
     
         49 . The method of  claim 48 , wherein the strain comprises two copies of the dsbC gene integrated into the chromosome and a plasmid comprising two copies of the FkpA gene operably linked to a promoter. 
     
     
         50 . The method of  claim 48 , wherein the strain comprises two copies of the dsbC gene and two copies of the FkpA gene integrated into the chromosome. 
     
     
         51 . The method of  claim 48 , wherein the strain is capable of a high growth rate. 
     
     
         52 . A bacterial cell free synthesis system for expressing biologically active proteins comprising:
 i) the cell free extract of  claim 47  having an active oxidative phosphorylation system, and comprising biologically functioning tRNA, amino acids and ribosomes necessary for cell free protein synthesis and wherein the exogenous protein chaperone was expressed in the bacterial strain at a level of at least 1 gm/liter of extract; and   ii) a nucleic acid encoding a protein of interest,   where said bacterial cell free synthesis system expresses a protein of interest to a concentration of at least about 100 mg/L.   
     
     
         53 . The bacterial cell free synthesis system of  claim 53 , wherein the cell free extract is prepared from the a bacterial stain comprising two copies of the dsbC gene integrated into the chromosome. 
     
     
         54 . The bacterial cell free synthesis system of  claim 53 , wherein the stain further comprises a plasmid comprising two copies of the FkpA gene operably linked to a promoter. 
     
     
         55 . The bacterial cell free synthesis system of  claim 53 , wherein the cell free extract is prepared from the a bacterial stain comprising two copies of the FkpA gene integrated into the chromosome. 
     
     
         56 . The bacterial cell free synthesis system of  claim 53 , wherein the cell free extract is prepared from the a bacterial stain comprising two copies of the dsbC gene and two copies of the FkpA gene integrated into the chromosome. 
     
     
         57 . A method of expressing properly folded, biologically active proteins in a bacterial cell free synthesis system comprising the steps of:
 i) incubating the bacterial cell free synthesis system of  claim 53  under conditions permitting the expression and proper folding of the protein of interest.