A bifunctional chimeric protein consisting of MutS and beta-galactosidase.
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 1l 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 beta-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 microl volumes after 120 and 15 min incubation, respectively.
Sachadyn
Paweł
P
Gdańsk University of Technology, Department of Microbiology, ul. Narutowicza 11/12, 80-952 Gdańsk, Poland. psach@altis.chem.pg.gda.pl
Stanisławska-Sachadyn
Anna
A
Kur
Józef
J
D020137:Base Pair Mismatch; D020029:Base Pairing; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016963:Thermus thermophilus / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D001616:beta-Galactosidase; D051719:MutS DNA Mismatch-Binding Protein