Production of DNA polymerase by recombinant pET-17b/Pfu-Pol and its optimization for PCR

Abstract

Polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology and biotechnology. Thermostable DNA polymerase is the key enzyme that catalyzes the amplification of DNA templates during PCR cycles. Although this enzyme has been produced worldwide, there is no reported cloning or production of polymerases in Egypt. In the current work, plasmid coding Pfu polymerase enzyme (pET-17b/Pfu-Pol) was transformed into E. coli Top10. The plasmid coding Pfu- polymerase was confirmed by restriction analysis using HindIII (5.5Kbp) and by double digestion with HindIII/XhoI (3.6 and 1.95Kbp). Furthermore, the confirmed plasmid was transformed into E. coli BL21 (DE3) pLysS cells for protein expression. Enzyme production was induced with 0.5mM IPTG and precipitated with ammonium sulfate. The SDS-PAGE analysis of the enzyme showed single protein band at 86KDa. Different conditions were assessed for optimization of the prepared enzyme including Mg ion concentration, the amount of used enzyme and the extension temperature. Taq enzyme showed more PCR potential activity using 4mM MgCl₂ with 0.5μl of the prepared enzyme in the final reaction mixture (25μl). Also, the enzyme was active when PCR was conducted at extension temperature 72º C. Moreover, the produced Pfu-polymerase showed similar enzyme activity compared to the commercially available polymerases.