High expression and identification of DNA mismatch repair gene mutS in Escherichia coli.
- Author:
Li-Jun BI
1
;
Ya-Feng ZHOU
;
Jiao-Yu DENG
;
Xian-En ZHANG
;
Cheng-Gang ZHANG
;
Anthony E G CASS
Author Information
1. Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
- Publication Type:Journal Article
- MeSH:
Adenosine Triphosphatases;
biosynthesis;
genetics;
isolation & purification;
Bacterial Proteins;
Base Pair Mismatch;
Chromatography, Affinity;
DNA;
metabolism;
DNA Repair;
DNA-Binding Proteins;
Escherichia coli Proteins;
biosynthesis;
genetics;
isolation & purification;
Magnesium;
pharmacology;
Molecular Weight;
MutS DNA Mismatch-Binding Protein;
Recombinant Proteins;
biosynthesis
- From:
Chinese Journal of Biotechnology
2002;18(5):536-540
- CountryChina
- Language:Chinese
-
Abstract:
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.
