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Effect of controlled redox potential and dissolved oxygen on the in vitro refolding of an E. coli alkaline phosphatase and chicken lysozyme

Paper ID Volume ID Publish Year Pages File Format Full-Text
17088 42640 2013 7 PDF Available
Title
Effect of controlled redox potential and dissolved oxygen on the in vitro refolding of an E. coli alkaline phosphatase and chicken lysozyme
Abstract

•For the first time the effect of controlling DOT or RP on protein folding is shown for two model proteins.•An improvement of protein refolding was observed at oxidizing values when RP and DOT control was done for both enzymes.•The results obtained in this manuscript are helpful to improve protein refolding by dilution.

The development of efficient purification strategies of recombinant active protein derived from inclusion bodies requires the knowledge of the effect of environmental variables, such as redox potential (RP) and dissolved oxygen tension (DOT), in order to control the protein folding process. However, that information is scarce and only few in vitro studies of the impact of such variables have been reported under constant controlled conditions. In this work, the effect of controlled RP and DOT on the refolding of E. coli alkaline phosphatase (AP) and chicken lysozyme (CL) enzymes were studied. Disulphide bonds of both enzymes were reduced in an instrumented vessel using 2-mercaptoethanol and nitrogen. In the latter case, guanidine hydrochloride was also used to denature the protein. Such conditions caused protein conformational changes, as determined by the intrinsic fluorescence spectra that correlated with a decrease on the activity in both cases. Reduced enzymes were then oxidized, under different constant and predetermined RP or DOT, by manipulating the gas composition in the vessel. Folding kinetics were followed as the recovery of enzyme activity. Results showed that the percentage of recovery and rate of increase of enzymatic activity directly depended on the RP and DOT. A higher folding efficiency was found under controlled DOT compared to controlled RP conditions. These results are useful for establishing protein folding strategies to improve the recovery of active protein from inclusion bodies.

Keywords
Alkaline phosphatase; Chicken lysozyme; Dissolved oxygen tension; Protein refolding; Redox potential
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Effect of controlled redox potential and dissolved oxygen on the in vitro refolding of an E. coli alkaline phosphatase and chicken lysozyme
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 52, Issues 6–7, 10 May 2013, Pages 312–318
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