Cloning,Expression,Purification,Characterization And Improvement Of An Archaeal DNA Polymerase

DNA polymerase is a key enzyme that is ubiquitous in living organisms for DNA duplication.With the development of molecular biology research,many DNA polymerases have been found and gradually applied to include PCR,Biological research including sitedirected mutagenesis and high-throughput sequencing.Among the many DNA polymerases,archaeal DNA polymerases from family B have good thermal stability,superior fidelity,and certain processability.The KOD DNA polymerase from archaeal Thermococcus kodakarensis studied in this paper belongs to this family,but most of the KOD DNA polymerase products on the market come from foreign companies,which are restricted by imports and patents,and the price is more expensive.Therefore,it is very important to develop a KOD DNA polymerase with excellent characteristics and price advantage.In this paper,we use Escherichia coli to express KOD DNA polymerase and characterize its biological activity;at the same time,a KOD DNA polymerase mutant was constructed by site-directed mutagenesis,and its biological activity was also characterized;we constructed the PCR complex enzyme system by adding helicase or chaperonin,the specificity,storage stability and damage repair of PCR were studied.The research results show as followed:The concentration of recombinant KOD DNA polymerase expressed in E.coli was reach up to 5.436 mg/m L,purity of more than 90%,5'-3' polymerase specific activity was 1.02×104 U/mg,with a good exonuclease activity from 3'-5',and can be well amplified with plasmid as template,the fidelity is about 1.60×10-5;the recombinant KOD DNA polymerase mutant L608 P did not obtain effective biological activity;in the process of PCR reaction,we added DNA helicase TK-Upf1 and TTE0604,the non-specific bands was decreased or disappeared,the specificity of PCR reaction was improved;the addition of chaperonin Cpk A and Cpk B can prolong the stability of KOD DNA polymerase at low temperature;at the same time,Cpk A and Cpk B can repair damaged and inactivated recombinant KOD DNA polymerase to a certain extent;Among them,the repair rate of Cpk A was 57.5% and the repair rate of Cpk B was 35.8%.The results of this study provide a laboratory basis and research ideas for the basic research and application development of KOD DNA polymerase.