Purification Of Recombinant DNA Polymerase In E.coli

PCR is one of the most important tools in molecular biology. DNA polymerase takes a critical role in the PCR technology. Taq DNA polymerase is the first one which is applied in the PCR. Pfu DNA polymerase, having 3'-5' exonuclease activity, is one of the DNA polymerases that have the highest fidelity. This study concentrated on extracting and purification of Taq and Pfu DNA polymerase in the recombinant strains.The expression plasmid of p-Taq was transformed in E. coli DH5a. Using the thermostable ability of Taq DNA polymerase, the protein was immerged in a 75℃ water-bath for 1h, and then obtained by the method of ammonium sulfate precipitation. The culture conditions of recombinant E.coli were studied. The optimal culture conditions were that: induced at OD₆₀₀=0.8 with 125mg/L IPTG, and grown at 37℃ for 9h. Then 50,000 unit of polymerase was obtained from 1L culture, with a specific activity of 10,000U/mg.The recombinant strain of Pfu DNA polymerase was constructed by transforming the expression plasmid pET-Pfu into BL21 (DE3 ) containing plasmid pLysS. During the purification of Pfu DNA polymerase, the cells were lysed by lysozyme, and a heat treatment step was performed to eliminate most contaminant proteins, using the thermostable ability of Pfu DNA polymerase. Then further purification of target protein by chromatography was studied, including cellulose phosphate (P11), CM-Sepharose CL-6B, DEAE-Sepharose FF, JK110, Sephacryl S-200, Sephadex G-100 and Sephadex G-75. Lactose, instead of IPTG, was used as the inducer, and the induce conditions were optimized. The optimized conditions were that: adding 1g/L lactose into the culture immediately after inoculation and the cells were harvest after grown for 8h. The purified protein was obtained by a heat-treatment at 75℃ for 20min and a JK110 chromatography and Sephadex G-75 gel filtration. In the PCR amplification, our purified polymerase had similar result to the commercial one. We obtained 3×10⁵U of purified Pfu DNA polymerase from 500ml culture, with a specific activity of 22,200U/mg and a concentration of 10U/(?)1.