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Refolding of hexameric porcine leucine aminopeptidase using a cationic detergent and dextrin-10 as artificial chaperones

Paper ID Volume ID Publish Year Pages File Format Full-Text
24242 43507 2009 7 PDF Available
Title
Refolding of hexameric porcine leucine aminopeptidase using a cationic detergent and dextrin-10 as artificial chaperones
Abstract

The application of artificial chaperones in biotechnology has been inspired by the mechanism of molecular chaperones like GroEL/GroES. It involves addition of a capturing detergent during dilution of the chaotropic reagent, that prevents protein aggregation, and finally, addition of a oligosaccharide that removes the detergent allowing the protein to refold. Here, guanidinium hydrochloride-denatured hexameric leucine aminopeptidase is shown to be efficiently refolded by using the cationic detergent cetyltrimethylammonium bromide and the linear polysaccharide dextrin-10 as artificial chaperones. The effect of these additives and the time dependence on the recovery of total enzymatic activity, kinetic parameters (KM, kcat), intrinsic steady-state tryptophan fluorescence and oligomeric structure is presented. The method described is very promising since 92% of fully active and correct folded LAP could be produced. Moreover, we showed that the stripping process is relatively slow, it allows the protein to refold almost entirely to its native state.

Keywords
LAP, leucine aminopeptidase; CTAB, cetyltrimethylammonium bromide; GuHCl, guanidinium hydrochloride; DTT, dithiotreitol; KM, Michaelis–Menten constant; kcat, turnover number; kobs, observed rate constant; SDS–PAGE, sodium dodecyl sulfate polyacrylamide ge
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Refolding of hexameric porcine leucine aminopeptidase using a cationic detergent and dextrin-10 as artificial chaperones
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 140, Issues 3–4, 25 March 2009, Pages 162–168
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