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The construction of bifunctional fusion proteins consisting of MutS and GFP

Paper ID Volume ID Publish Year Pages File Format Full-Text
25726 43594 2006 10 PDF Available
Title
The construction of bifunctional fusion proteins consisting of MutS and GFP
Abstract

MutS as a mismatch binding protein is a promising tool for SNP detection. Green fluorescent protein (GFP) is known as an excellent reporter domain. We constructed chimeric proteins consisting of MutS from Thermus thermophilus and GFPuv from Aequorea victoria by cloning the GFPuv gene into the plasmid vectors carrying the mutS gene. The GFPuv domain fused to the N-terminus of MutS (histag-GFP-MutS) exhibited the same level of green fluorescence as free GFPuv. To obtain the fluorescing histag-GFP-MutS protein the expression at 30 °C was required, while free GFPuv fluoresces when expressed both at 30 and 37 °C. The chimeric protein where the GFPuv domain was fused to the C-terminus of MutS exhibited much weaker green fluorescence (20–25% compared with those of histag-GFP-MutS or free GFPuv). The insertion of (ProGly)5 peptide linker between the MutS and GFP domains resulted in no significant improvement in GFP fluorescence. No shifts in the excitation and emission spectra have been observed for the GFP domain in the fusion proteins. The fusion proteins with GFP at the N- and C-terminus of MutS recognised DNA mismatches similarly like T. thermophilus MutS. The fluorescent proteins recognising DNA mismatches could be useful for SNP scanning or intracellular DNA analysis.The fusion proteins around 125 kDa were efficiently expressed in E. coli and purified in milligram amounts using metal chellate affinity chromatography.

Keywords
BSA, bovine serum albumine; PAGE, polyacrylamide gel electrophoresis; rbs, ribosomal binding site; SNP, single nucleotide polymorphismMutS; GFP; Fusion; Chimeric protein; SNP; Mutation detection
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The construction of bifunctional fusion proteins consisting of MutS and GFP
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 121, Issue 2, 24 January 2006, Pages 134–143
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