RECOMBINANT PROTEINS CAN BE ISOLATED FROM ESCHERICHIA-COLI-CELLS BY REPEATED CYCLES OF FREEZING AND THAWING

Repeated cycles of freezing and thawing are sufficient to separate hig hly expressed recombinant proteins away from the cellular milieu of E. coli. Freezing and thawing liberates recombinant proteins from the ba cterial cytoplasm, but does not release the bulk of endogenous E. coli proteins. Furthermore, protein secretion is not required. Fractionati on of overexpressed proteins by freeze/thaw treatment does not depend on the identity of the recombinant protein and has been observed for t hirty-five different recombinant proteins expressed in E. coli. These include proteins originally found in plant, animal or microbial source s, as well as several proteins designed de novo. Freezing and thawing typically yields similar to 50% of the recombinant protein in relative ly pure form. Thus the freeze/thaw treatment can be utilized as a gene ral method for the isolation of recombinant proteins from E. coli.