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A bifunctional chimeric protein consisting of MutS and beta-galactosidase

Paper ID Volume ID Publish Year Pages File Format Full-Text
25543 43580 2007 6 PDF Available
Title
A bifunctional chimeric protein consisting of MutS and beta-galactosidase
Abstract

A bifunctional protein consisting of MutS, a mismatch binding protein and a beta-galactosidase reporter domain has been constructed. The fusion of beta-galactosidase to the MutS C-terminus was obtained by cloning the Escherichia coli lacZ gene encoding beta-galactosidase into a plasmid vector carrying the Thermus thermophilus mutS gene. Milligram amounts of this huge chimeric protein (217 kDa monomer) were purified from 1 l of overexpressing E. coli cells using metal-chelate affinity chromatography. The mismatch binding properties of the fusion protein were confirmed by DNA mobility shift assay in polyacrylamide gels. Binding to biotinylated mismatched DNA immobilized on streptavidin microplates followed by colorimetric reaction with X-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside), demonstrated both mismatch recognition and beta-galactosidase activity of the chimeric protein. The activity of beta-galactosidase domain of the fusion was similar to that of the native enzyme. A colorimetric assay for β-galactosidase activity using X-Gal supplemented with NBT (nitro blue tetrazolium) allowed detection of 50 and 500 fmol of the chimeric protein with naked eye in 45 μl volumes after 120 and 15 min incubation, respectively.

Keywords
BSA, bovine serum albumin; IPTG, isopropyl-beta-d-thiogalactopyranoside; X-Gal, 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside; NBT, nitro blue tetrazolium; PBS, phosphate buffered salineMutS; Beta-galactosidase; Fusion; Chimeric protein; Mutation de
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A bifunctional chimeric protein consisting of MutS and beta-galactosidase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 127, Issue 2, 1 January 2007, Pages 229–234
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