ISSN:
1476-5535
Keywords:
recombinant isopenicillin N synthase
;
expression
;
penicillin
;
Streptomyces clavuligerus
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01570004
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