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Increasing the refolding efficiency in vitro by site-directed mutagenesis of Cys383 in rat procarboxypeptidase B

Paper ID Volume ID Publish Year Pages File Format Full-Text
17397 42666 2011 7 PDF Available
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 kcat/Km 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.

Keywords
Procarboxypeptidase B; Mutation; Cysteine; Refolding efficiency; Property
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Increasing the refolding efficiency in vitro by site-directed mutagenesis of Cys383 in rat procarboxypeptidase B
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 49, Issue 2, 10 July 2011, Pages 139–145
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us