title: Purification and characterization of recombinant Streptomyces clavuligerus isopenicillin N synthase produced in Escherichia coli.
abstract: Recombinant isopenicillin N synthase from Streptomyces clavuligerus was produced in the form of inactive inclusion bodies in Escherichia 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 degrees 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.
authors.affiliation: Department of Biological Sciences, University of Alberta, Edmonton, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Durairaj
authors.forename: S E
authors.initials: SE
authors.lastname: Jensen
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D002479:Inclusion Bodies / Q000378:metabolism; D019751:Intramolecular Transferases; D007535:Isomerases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D007535:Isomerases; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase