Single-step purifications of His6-MutH, His6-MutL and His6-MutS repair proteins of escherichia coli K-12.
The MutS, MutH and MutL proteins mediate methyl-directed-mismatch (MDM) repair in Escherichia coli and Salmonella typhimurium. These proteins have been developed into powerful tools for screening genomes for polymorphisms and detecting and localizing mutations. In an ongoing study of the regulation of MDM repair, we developed one-step schemes to purify the E. coli MutS, MutH and MutL proteins fused to a polyhistidine (His6) affinity tag. The E. coli K-12 mutS+, mutH+ and mutL+ genes were cloned from the Clarke-Carbon plasmid or Kohara-phage library into expression vector pET-15b, which allows fusion to the His6 affinity tag. Each of the resulting recombinant plasmids complemented the corresponding mutHLS mutation in the Cupples-Miller CC106 mutator tester strain, indicating that the His6-MutHLS fusion proteins were individually functional in vivo. The His6-MutHLS proteins were separately purified by variations of batch binding to Ni(2+)-chelation affinity resin. The yield of purified His6-MutHLS proteins from these procedures was 0.4-0.6 mg from 40 mL of induced culture. The binding properties of one-step-purified His6-MutS protein were characterized further. His6-MutS exhibited the same mismatch-binding activity and specificity as native MutS in side-by-side bandshift assays. These constructs and purification methods should be useful to laboratories wishing to apply or develop MutS-mismatch mapping and to other applications or studies of the E. coli MutHLS repair proteins.
Feng
G
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University of Texas, Houston Medical School, USA.
Winkler
M E
ME
D000251:Adenosine Triphosphatases; D000345:Affinity Labels; D001426:Bacterial Proteins / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D004706:Endodeoxyribonucleases / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006639:Histidine; D008969:Molecular Sequence Data; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; D009154:Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding
D000345:Affinity Labels; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D006639:Histidine; D004706:Endodeoxyribonucleases; C059682:methyl-directed mismatch repair protein, E coli; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli; D045643:DNA Repair Enzymes