Biophysical factors affecting the expression of isopenicillin N synthase isozymes in Escherichia coli.
The influence of biophysical factors over the production of isozymes of isopenicillin N synthase (IPNS) from fungal and bacterial sources in Escherichia coli was examined. The effect of these parameters on the expression of Cephalosporium acremonium IPNS was determined by using two expression vectors containing the lac- or the T7-promoter. Under the lac-promoter, IPNS expressed in E. coli reached 5-15% of total soluble proteins and under the T7 promoter, the level reached approximately 50% of total soluble proteins. Furthermore the high level of expression did not affect aggregation of IPNS proteins. Analysis of the hydrophobicity of the C. acremonium IPNS and site-specific mutant enzymes relative to other IPNS isozymes suggested that proteins of low gravy scores (< or = -0.515) tend to be soluble whereas higher gravy scores (> or = -0.512) form aggregates. Neither the GC content of the gene nor the relative percentage proline content of the protein affected the expression of the IPNS isozymes in E. coli.
Tiow-Suan
S
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Department of Microbiology, Faculty of Medicine, National University of Singapore.
Tan
D S
DS
D000164:Acremonium / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics*; D011392:Proline / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D013499:Surface Properties
D007527:Isoenzymes; D011994:Recombinant Proteins; D004247:DNA; D011392:Proline; D010088:Oxidoreductases; C038370:isopenicillin N synthetase