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

A stable expression plasmid has been developed to overproduce the Escherichia coli GroES and GroEL molecular chaperones in large-scale cultures. This was achieved by cloning the groE operon under the transcriptional control of a bacteriophage T7 promoter to achieve regulated expression. Isopropyl-beta-D-thiogalactopyranoside (IPTG) induction of a lacUV5 regulated chromosomal copy of T7 gene 1, encoding viral RNA polymerase, resulted in high-level expression of the groE operon from a multicopy plasmid. Induced cells harboring the pT7groE expression plasmid accumulated GroEL to levels of 30% total cell protein, and GroES to 4-5%. Both overproduced proteins were recovered primarily from the soluble fraction of lysed cells. The T7 expression plasmid was significantly more stable than other groE expression plasmids tested during scale-up experiments, and could be used successfully for large-volume cultures of up to 200 l. Strain stability was greatly improved, compared to rich media, when cells were grown in a supplemented minimal medium.