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.
Kalbach
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Molecular Biology Division, E. I. DuPont de Nemours and Company, Wilmington, Delaware 19880-0402.
Gatenby
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D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics; D018835:Chaperonin 10; D018834:Chaperonin 60; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009876:Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D007544:Isopropyl Thiogalactoside; D012321:DNA-Directed RNA Polymerases