Molecular Cloning and Chartacterization DNA Polymerase I from thermophilic Geobacillus SBS 4S strain

Thermostable DNA polymerases are extensively used in biotechnology and life science applications. DNA Polymerase I was isolated from a hyperthermophile bacteria Geobacillus SBS 4S. Primers were designed using the template sequence of DNA Polymerase I gene of Geobacillus kaustophilus HTA26 strain. Nco 1 and Hind III sites were introduced on the forward and reverse primers respectively. Polymerase I gene of 2.6 Kb was cloned in pTZ57/ RT vector. Cloned gene of Polymerase I was restricted with Nco 1 and Bam H1, and ligated to pET 22b vector. Nco 1 site was used to insert twenty two N-terminal aminoacids (pelB) leader sequence at the start of the gene, which lead the recombinant protein in the periplasmic space, which increases the half life of recombinant protein. pelB fused with DNA Polymerase I produces soluble protein, which was detected after sonication. Sequencing shows that DNA polymerase I consists of 2499 bp with encodes for 832 amino acids, showed 99 % similarity with Geobacillus Kaustophillus . The expression of pelB fused DNA Polymerase I was optimized at different concentrations of IPTG and lactose. Highest expression was observed with 0.5mM IPTG and 20mM lactose. After Harvesting and sonication of BL21 codon plus cells, Polymerase I was produced in the soluble fraction. The supernatant containing the protein of interest, was separated after centrifugation at 10,000 rpm for 20 min. The protein was purified by ammonium sulphate precipitation and cation exchange column. The activity of purified DNA Polymerase I was checked by PCR reaction.