Open AccessHigh level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces , and recovery of active enzyme from inclusion bodies
Author(s) -
Landman Orna,
Shiffman Dov,
AvGay Yosef,
Aharonowitz Yair,
Cohen Gerald
Publication year - 1991
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1991.tb04603.x
Subject(s) - escherichia coli , flavobacterium , streptomyces , enzyme , gene , biology , microbiology and biotechnology , streptomycetaceae , biochemistry , genetics , actinomycetales , bacteria , pseudomonas
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.