Construction and purification of his6-Thermus thermophilus MutS protein.
The mutS gene from the thermophilic bacterium Thermus thermophilus was PCR amplified, cloned, and expressed in Escherichia coli. The recombinant MutS protein containing an oligohistidine domain at the N-terminus was purified in a single step by Ni(2+) affinity chromatography to apparent homogeneity. The mismatch recognition properties of the his(6)-tagged MutS protein were confirmed by DNA protection against exonuclease digestion and retardation assays. The results of analytical gel filtration indicate that the predominant form of T. thermophilus MutS at micromolar concentrations is a tetramer.
Stanisławska-Sachadyn
Anna
A
Technical University of Gdańsk, Department of Microbiology, ul. Narutowicza 11/12, Gdansk 80-952, Poland.
Sachadyn
Paweł
P
Jedrzejczak
Robert
R
Kur
Józef
J
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020137:Base Pair Mismatch / Q000235:genetics; D001483:Base Sequence; D002850:Chromatography, Gel; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D005822:Genetic Vectors; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013194:Staining and Labeling; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D004247:DNA; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli