[High expression and identification of DNA mismatch repair gene mutS in Escherichia coli].
DNA mismatch repair gene mutS (2.56 kb) was PCR modified and cloned into a secretive prokaryotic expression vector pET32a (+) which carries a N-terminal His.tag + and thioredoxin sequence. MutS protein was expressed with high level after IPTG induction using the strain E. coli AD494(DE3). SDS-PAGE revealed that the expected protein with a molecular weight of 108 kD which is about 35% of the total bacterial proteins is almost soluble. The expected protein was purified directly by immobilized metal (Ni2+) chelation affinity chromatography and the purity is over 90%. MutS protein activity verified using mismatch DNA showed that the expression product can recognize and bind to base-pair mismatch specifically.
Bi
Li-Jun
LJ
Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Zhou
Ya-Feng
YF
Deng
Jiao-Yu
JY
Zhang
Xian-En
XE
Zhang
Cheng-Gang
CG
Cass
Anthony E G
AE
D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins; D020137:Base Pair Mismatch; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D051719:MutS DNA Mismatch-Binding Protein; D011994:Recombinant Proteins / Q000096:biosynthesis*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D004247:DNA; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli; D008274:Magnesium