title: Increasing the refolding efficiency in vitro by site-directed mutagenesis of Cys383 in rat procarboxypeptidase B.
abstract: This study examines a novel method to reduce the probability of disulfide mismatches during the refolding process by the replacement of cysteines within a protein. Specifically, Cys383 of recombinant rat procarboxypeptidase B was replaced by other amino acids to increase the refolding efficiency in vitro. Mutants C383G, C383A and C383S could refold successfully, but mutants C383R, C383E, C383L and C383Y failed to refold correctly. Compared with wild type, the refolding efficiencies of mutants C383G and C383A were enhanced. The Cys383 mutations changed some of the properties of rat carboxypeptidase B. Mutants C383G, C383A had higher k(cat)/K(m) values which indicated increased catalytic abilities. And both had higher thermal stability. pH had different effects on the activities and stabilities of the mutant and wild type proteins. The studies suggested that mutating Cys383 of rat procarboxypeptidase B could improve the renaturation process by increasing the refolding efficiency. This new method could be taken as a new attempt to improve the refolding efficiency of other recombinant proteins containing disulfide bonds that are expressed as inclusion bodies. While the results also claimed that the potential effects of the substituted amino acid on the protein itself should be seriously considered in addition to its ability to reduce the probability of disulfide mismatches.
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China. lisuxia@ecust.edu.cn
authors: Suxia
authors: S
authors: Li
authors: Luosheng
authors: L
authors: Zhang
authors: Qian
authors: Q
authors: Wu
authors: Aijie
authors: A
authors: Xin
authors: Jian
authors: J
authors: Zhao
authors: Liqiang
authors: L
authors: Fan
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D043424:Carboxypeptidase B / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; D050505:Mutant Proteins; D011994:Recombinant Proteins; D043424:Carboxypeptidase B; D003545:Cysteine