caa a22 4500 477106056 CHVBK 20180405111553.0 cr unu---uuuuu 170330e19960301xx s 000 0 eng 10.1007/BF01570004 doi (NATIONALLICENCE)springer-10.1007/BF01570004 Purification and characterization of recombinant Streptomyces clavuligerus isopenicillin N synthase produced in Escherichia coli [Elektronische Daten] [M Durairaj, S Jensen] 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. Society for Industrial Microbiology, 1996 recombinant isopenicillin N synthase nationallicence expression nationallicence penicillin nationallicence Streptomyces clavuligerus nationallicence Durairaj M. Department of Biological Sciences, University of Alberta, T6G 2E9, Edmonton, AB, Canada aut Jensen S. Department of Biological Sciences, University of Alberta, T6G 2E9, Edmonton, AB, Canada aut Journal of Industrial Microbiology Springer-Verlag 16/3(1996-03-01), 197-203 0169-4146 16:3<197 1996 16 10295 https://doi.org/10.1007/BF01570004 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF01570004 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Durairaj M. Department of Biological Sciences, University of Alberta, T6G 2E9, Edmonton, AB, Canada aut NATIONALLICENCE 700 1- Jensen S. Department of Biological Sciences, University of Alberta, T6G 2E9, Edmonton, AB, Canada aut NATIONALLICENCE 773 0- Journal of Industrial Microbiology Springer-Verlag 16/3(1996-03-01), 197-203 0169-4146 16:3<197 1996 16 10295 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer