Abstract
We have successfully expressed and observed secretion of the Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) using the Pichia pastoris expression system. Two clones having multiple copies of the expression cassette were selected and used for protein-expression analysis. SDS-PAGE showed efficient expression and secretion of the bacterial recombinant DAOCS. The highest yield (120 microg/mL) was obtained when expression was induced with 2% methanol. Free and immobilized protein were assayed for biological activity and found to expand penicillin N (its natural substrate) and penicillin G to deacetoxycephalosporin C (DAOC) and deacetoxycephalosporin G (DAOG), respectively.
Copyright 2001 John Wiley & Sons, Inc.
MeSH terms
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Cephalosporins / biosynthesis
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Clone Cells
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Culture Media / analysis
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Electroporation
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Gene Expression
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Gene Transfer Techniques
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Hydrogen-Ion Concentration
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Intramolecular Transferases / biosynthesis*
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Methanol / pharmacology*
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Penicillin G / metabolism
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Penicillin-Binding Proteins*
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Penicillins / metabolism
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Periplasm / chemistry
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Pichia / genetics*
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Pichia / metabolism
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Plasmids
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / genetics
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Solvents / pharmacology
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Streptomyces / metabolism*
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Substrate Specificity
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Time Factors
Substances
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Cephalosporins
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Culture Media
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Penicillin-Binding Proteins
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Penicillins
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Recombinant Proteins
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Solvents
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deacetoxycephalosporin G
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Intramolecular Transferases
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deacetoxycephalosporin C synthetase
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penicillin N
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Penicillin G
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Methanol