Purification and characterization of recombinantStreptomyces clavuligerus isopenicillin N synthase produced inEscherichia coli |
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Authors: | M Durairaj Dr S E Jensen |
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Institution: | (1) Department of Biological Sciences, University of Alberta, T6G 2E9 Edmonton, AB, Canada |
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Abstract: | Recombinant isopenicillin N synthase fromStreptomyces clavuligerus was produced in the form of inactive inclusion bodies inEscherichia coli. These inclusion bodies were solubilized by treatment with 5 M urea under reducing conditions. Optimization of refolding conditions to recover active isopenicillin N synthase indicated that a dialysis procedure carried out at a protein concentration of about 1.0 mg ml–1 gave maximal recovery of active isopenicillin N synthase. Solubilized isopenicillin N synthase of more than 95% purity was obtained by passing this material through a DEAE-Trisacryl ion exchange column. Expression studies conducted at different temperatures indicated that isopenicillin N synthase was produced predominantly in a soluble, active form when expression was conducted at 20°C, and accounted for about 20% of the total soluble protein. This high-level production facilitated the purification of soluble isopenicillin N synthase to near homogeneity in four steps. Characterization of the purified soluble and solubilized isopenicillin N synthase revealed that they are very similar. |
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Keywords: | recombinant isopenicillin N synthase expression penicillin Streptomyces clavuligerus |
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