STABLE EXPRESSION PLASMID FOR HIGH-LEVEL PRODUCTION OF GROE MOLECULARCHAPERONES IN LARGE-SCALE CULTURES

A stable expression plasmid has been developed to overproduce the Esch erichia coli GroES and GroEL molecular chaperones in large-scale cultu res. This was achieved by cloning the groE operon under the transcript ional control of a bacteriophage T7 promoter to achieve regulated expr ession. Isopropyl-beta-D-thiogalactopyranoside (IPTG) induction of a l acUV5 regulated chromosomal copy of T7 gene 1, encoding viral RNA poly merase, resulted in high-level expression of the groE operon from a mu lticopy plasmid. Induced cells harboring the pT7groE expression plasmi d accumulated GroEL to levels of 30% total cell protein, and GroES to 4-5%. Both overproduced proteins were recovered primarily from the sol uble fraction of lysed cells. The T7 expression plasmid was significan tly more stable than other groE expression plasmids tested during scal e-up experiments, and could be used successfully for large-volume cult ures of up to 200 l. Strain stability was greatly improved, compared t o rich media, when cells were grown in a supplemented minimal medium.