The construction of bifunctional fusion proteins consisting of MutS and GFP.
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 degrees C was required, while free GFPuv fluoresces when expressed both at 30 and 37 degrees 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.
Stanisławska-Sachadyn
Anna
A
Gdańsk University of Technology, Department of Microbiology, ul. Narutowicza 11/12, 80-952 Gdańsk, Poland.
Sachadyn
Paweł
P
Ihle
Karolina
K
Sydorczuk
Cezary
C
Wiejacha
Katarzyna
K
Kur
Józef
J
D020137:Base Pair Mismatch; D004252:DNA Mutational Analysis / Q000379:methods; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics*; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D016963:Thermus thermophilus / Q000737:chemistry / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D051719:MutS DNA Mismatch-Binding Protein