caa a22 4500 475744268 CHVBK 20180406123508.0 cr unu---uuuuu 170329e20000501xx s 000 0 eng 10.1023/A:1007043312464 doi (NATIONALLICENCE)springer-10.1023/A:1007043312464 Analysis of the Disulfide Bonding Pattern between Domain Sequences of Recombinant Prochymosin Solubilized from Inclusion Bodies [Elektronische Daten] [Chongjuan Wei, Hongjie Chen, Yuying Zhang, Kaiyu Yang] Prochymosin contains three disulfide bonds linking Cys45 to Cys50, Cys206 to Cys210, and Cys250 to Cys283. To analyze the disulfide bonding pattern between domain sequences in the recombinant prochymosin molecule solubilized from inclusion bodies by 8 M urea (designated as solubilized prochymosin), a simple peptide mapping method was established. This process consists of thiol alkylation, cleavage with cyanogen bromide, diagonal electrophoresis on polyacrylamide gel, and N-terminal sequencing. By using this procedure it was found that Cys45 and Cys50 located in the N-terminal domain are not mispaired with the cysteine residues, located in the C-terminal domain, in the solubilized wild-type prochymosin and its mutants. This result implies that Cys45 and Cys50, the partners of a native disulfide, are restricted in some ordered structures existing in inclusion bodies and remaining after solubilization. These native structural elements act as folding nuclei to initiate and facilitate correct refolding. The strategy of preserving the native-like structures including native disulfide in the solubilized inclusion bodies to enhance renaturation efficiency may be applicable to other recombinant proteins. Plenum Publishing Corporation, 2000 Prochymosin domain nationallicence disulfide mapping nationallicence diagonal electrophoresis nationallicence refolding nationallicence inclusion bodies nationallicence Wei Chongjuan Department of Enzymology, Institute of Microbiology, Chinese Academy of Sciences, 100080, Beijing, China aut Chen Hongjie Department of Enzymology, Institute of Microbiology, Chinese Academy of Sciences, 100080, Beijing, China aut Zhang Yuying Department of Enzymology, Institute of Microbiology, Chinese Academy of Sciences, 100080, Beijing, China aut Yang Kaiyu Department of Enzymology, Institute of Microbiology, Chinese Academy of Sciences, 100080, Beijing, China aut Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/4(2000-05-01), 277-284 0277-8033 19:4<277 2000 19 10930 https://doi.org/10.1023/A:1007043312464 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1007043312464 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Wei Chongjuan Department of Enzymology, Institute of Microbiology, Chinese Academy of Sciences, 100080, Beijing, China aut NATIONALLICENCE 700 1- Chen Hongjie Department of Enzymology, Institute of Microbiology, Chinese Academy of Sciences, 100080, Beijing, China aut NATIONALLICENCE 700 1- Zhang Yuying Department of Enzymology, Institute of Microbiology, Chinese Academy of Sciences, 100080, Beijing, China aut NATIONALLICENCE 700 1- Yang Kaiyu Department of Enzymology, Institute of Microbiology, Chinese Academy of Sciences, 100080, Beijing, China aut NATIONALLICENCE 773 0- Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/4(2000-05-01), 277-284 0277-8033 19:4<277 2000 19 10930 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer