High level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces, and recovery of active enzyme from inclusion bodies.
A T7 promoter-based vector was used to express the isopenicillin N synthase (IPNS) genes of Flavobacterium sp. 12,154 and Streptomyces jumonjinensis in Escherichia coli. Most of the IPNS synthesized at 37 degrees C, and representing some 22% and 51% of the total cell protein respectively, occurred in an insoluble, enzymatically inactive form. Active IPNS was recovered in a rapid and simple two-step procedure in which the insoluble material was first denatured in 5 M urea and then refolded by passing the solubilized IPNS through a G-25 Sephadex sizing column. Further chromatography on DEAE-Sepharose resulted in highly active IPNS preparations. This procedure was found to be well suited for scaling up to produce large amounts of IPNS.
Landman
O
O
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel.
Shiffman
D
D
Av-Gay
Y
Y
Aharonowitz
Y
Y
Cohen
G
G
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005417:Flavobacterium / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D002479:Inclusion Bodies / Q000201:enzymology*; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification; D013302:Streptomyces / Q000235:genetics*
D010088:Oxidoreductases; C038370:isopenicillin N synthetase