High-level expression of the Streptomyces clavuligerus isopenicillin N synthase gene in Escherichia coli

The pcbC gene, which encodes isopenicillin N synthase (IPNS), was subcloned from Streptomyces clavuligerus into Escherichia coli by using the pT7 series of plasmid vectors. The polymerase chain reaction was used to introduce an NdeI site at the translation initiation codon of pcbC, allowing the gene to be inserted behind an E. coli type of ribosome binding site. This construction directed high-level expression of IPNS, but the IPNS was in an inactive form in inclusion bodies. Active IPNS was recovered by solubilizing and renaturing the protein.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  D. Shiffman,et al.  High level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces, and recovery of active enzyme from inclusion bodies. , 1991, FEMS microbiology letters.

[3]  M. Wright,et al.  High-level soluble expression of isopenicillin N synthase isozymes in E. coli , 1991 .

[4]  C. Kurland.  Codon bias and gene expression , 1991, FEBS letters.

[5]  K. Chater,et al.  TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[6]  V. J. Chen,et al.  Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans , 1988, Journal of bacteriology.

[7]  J. Kane,et al.  Formation of recombinant protein inclusion bodies in Escherichia coli , 1988 .

[8]  A. Smallwood,et al.  Heterologous expression in Escherichia coli : effects of alterations in the sequence 5′ to the initiation codon , 1988 .

[9]  D. Westlake,et al.  Purification of isopenicillin N synthetase from Streptomyces clavuligerus. , 1986, Canadian journal of microbiology.

[10]  P. Skatrud,et al.  Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium , 1985, Nature.

[11]  C. Richardson,et al.  A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[12]  A. Demain,et al.  Enzymatic approach to syntheses of unnatural beta-lactams. , 1984, Science.

[13]  P. Model,et al.  Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid , 1984, Journal of bacteriology.

[14]  G. Stormo,et al.  lacZ translation initiation mutations. , 1984, Journal of molecular biology.

[15]  D. Westlake,et al.  High performance liquid chromatographic assay of cyclization activity in cell-free systems from Streptomyces clavuligerus. , 1982, The Journal of antibiotics.

[16]  D. Westlake,et al.  Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus. , 1988, Gene.

[17]  D. Ramón,et al.  Cloning and characterization of the isopenicillin N synthetase gene mediating the formation of the beta-lactam ring in Aspergillus nidulans. , 1987, Gene.

[18]  P. Skatrud,et al.  Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum. , 1986, Gene.

[19]  Y. Fujisawa,et al.  Biosynthesis of cephalosporins. , 1976, Advances in applied microbiology.

[20]  今中 宏,et al.  Penicillium chrysogenum に対するγ線照射について , 1956 .