ABSTRACT
The developments of recombinant DNA technology and structural biology make it possible to modify enzyme in molecular level. Scientists show growing interests in the evolution or functional fusion of enzymes. Recent advances and applications of the molecular enzyme engineering are reviewed and discussed in this article.
Subject(s)
Enzymes , Genetics , Protein Engineering , Methods , Recombinant Fusion Proteins , Genetics , Research DesignABSTRACT
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.