EXTRACTIVE CULTIVATION OF RECOMBINANT ESCHERICHIA-COLI USING AQUEOUS 2-PHASE SYSTEMS FOR PRODUCTION AND SEPARATION OF INTRACELLULAR HEAT-SHOCK PROTEINS

The extractive cultivation of recombinant Escherichia coli cells to pr oduce, release, and separate heat shock proteins (HSPs; GroEL and GroE S) using poly(ethylene glycol) (PEG)/dextran (Dex) aqueous two-phase s ystems was developed. The growth rate of E. coli OW10/pND5 cells in th e PEG/Dex two-phase media was almost the same value as that in the con trol media. The addition of 0.1 M potassium phosphate salts (KPi) incr eased the productivity of HSPs with keeping the growth rate of E. coli cells relatively high. The partition coefficients of HSPs were improv ed to greater values when phosphate salts were added at a concentratio n of more than 0.1 M. As a result, PEG/Dex systems supplemented with 0 .1 M KPi were found to be the optimized two-phase systems for the extr active cultivation of E. coli cells. In the systems, the HSPs were sel ectively partitioned to the top phase while cells occupied the bottom phase and the interface between the two phases. This integrated proces s was extended to a semicontinuous operating mode, where the top phase containing the HSPs was recovered following intermittent heating and ultrasonic irradiation. The bottom phase containing cells and cell deb ris was recycled together with new top phase solution to repeat produc tion and recovery of HSPs.