PURIFICATION AND CHARACTERIZATION OF RECOMBINANT STREPTOMYCES-CLAVULIGERUS ISOPENICILLIN-N-SYNTHASE PRODUCED IN ESCHERICHIA-COLI

Recombinant isopenicillin N synthase from Streptomyces clavuligerus wa s produced in the form of inactive inclusion bodies in Escherichia col i. These inclusion bodies were solubilized by treatment with 5 M urea under reducing conditions. Optimization of refolding conditions to rec over active isopenicillin N synthase indicated that a dialysis procedu re carried out at a protein concentration of about 1.0 mg ml(-1) gave maximal recovery of active isopenicillin N synthase. Solubilized isope nicillin N synthase of more than 95% purity was obtained by passing th is material through a DEAE-Trisacryl ion exchange column. Expression s tudies conducted at different temperatures indicated that isopenicilli n 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 homogenei ty in four steps. Characterization of the purified soluble and solubil ized isopenicillin N synthase revealed that they are very similar.