TY - JOUR
T1 - High level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces, and recovery of active enzyme from inclusion bodies
AU - Landman, Orna
AU - Shiffman, Dov
AU - Av-Gay, Yosef
AU - Aharonowitz, Yair
AU - Cohen, Gerald
N1 - Funding Information:
The research was supported by a joint grant from the National Council for Research and Development Israel and GBF, F.R.G.
PY - 1991/12/1
Y1 - 1991/12/1
N2 - 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.
AB - 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.
KW - Flavobacterium
KW - IPNS
KW - Isopenicillin N synthase
KW - Streptomyces jumonjinensis
KW - β-Lactam antibiotics
UR - http://www.scopus.com/inward/record.url?scp=0026289302&partnerID=8YFLogxK
U2 - 10.1111/j.1574-6968.1991.tb04603.x
DO - 10.1111/j.1574-6968.1991.tb04603.x
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AN - SCOPUS:0026289302
SN - 0378-1097
VL - 84
SP - 239
EP - 244
JO - FEMS Microbiology Letters
JF - FEMS Microbiology Letters
IS - 3
ER -