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अमूर्त

PCR-based Gene Synthesis, Cloning, Expression, Purification and Characterization of Bst DNA Polymerase in E. coli Cells

Malarveli Suppan, Shaharum Shamsuddin, Asma Ismail and Venugopal Balakrishnan

Gene synthesis is a technique for modifying genes for studying gene function, structure and expression. There are several PCR based strategies and non-PCR based strategies for gene synthesis. Here we perform the 2 step gene synthesis combining assembly PCR and sequential overlap extension PCR strategies to synthesize the full length of Bst DNA polymerase (2,648bp) and express the protein in E. coli cells. The full length Bst DNA polymerase was divided into 5 short DNA Fragments and the oligonucloetides were designed for the entire sequence each with 40-45 mers. In step 1, the oligonuleotides of each fragment were assembled and then the gene fragment was amplified separately through assembly PCR method. In step 2, sequentially the two adjacent fragments were assembled through overlap extension PCR at a time until the full length gene completely synthesized and finally cloned into pCR®2.1-TOPO vector. Then the full length Bst DNA pol gene was subcloned into pET28a(+) expression vector and finally expressed in BL21(DE3) E. coli cells. The purified protein was identified by MALDITOF analysis. The polymerization activity of the recombinant Bst DNA polymerase was compared with commercial enzyme. The 98kDA recombinant Bst DNA polymerase succeed in amplification of 100bp DNA fragment via helicase dependent amplification (HDA).