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High level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces , and recovery of active enzyme from inclusion bodies

1991; Oxford University Press; Volume: 84; Issue: 3 Linguagem: Inglês

10.1111/j.1574-6968.1991.tb04603.x

1574-6968

Orna Landman, Dov Shiffman, Yossef Av‐Gay, Yair Aharonowitz, Gerald Cohen,

Plant-Microbe Interactions and Immunity

A T7 promoter-based vector was used to express the isopenicillin N synthase (IPNS) genes of Flavobacterium sp. 12,154 and Streptomyces jumomjinensis in Escherichia coli. Most of the IPNS synthesized at 37°C, and representing some 22% and 51% of the total cell protein respectively, occured 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.