Cloning of DNA Polymerase-1 Gene from Thermophilic Bacillus Licheniformis Strain NWMF1 into an E.coli Expression System

Abstract

DNA polymerase, catalyze template directed synthesis of DNA from nucleotide triphosphate. Thermostable DNA polymerase-ǀ (DNAP-1) has been a common reagent in molecular biology because of its use in DNA amplification and DNA sequencing by PCR. DNAP-1 produced in moderate thermophiles such as Bacillus species may not be suitable for PCR, However, moderately thermophilic DNAP-1 from Bacillus has been used in molecular biology techniques such as loop mediated isothermal amplification. It is a low cost alternative to detect certain infectious diseases such as tuberculosis, malaria and can be applied in low/middle income countries. The objective of the study was isolation and cloning of DNAP-1 gene from native thermophilic bacterium, Bacillus licheniformis strain NWMF1 and over-expression by using expression host E. coli BL21(DE3)pLysS. A gram ve endospore forming thermophilic bacterium was isolated from soil near the hot-water springs at Polonnaruwa, Sri Lanka. The identification of Bacillus licheniformis strain NWMF1 was carried out using morphological tests and 16s r.RNA gene sequence analysis. Initially the gene was cloned into pGEMT-easy vector and transformed into E. coli JM109 followed by sequence confirmation and protein blast analysis by NCBI. Thereafter the DNAP-1 gene re-cloned into PET28a vector and transformed into E. coli BL21(DE3)pLysS expression host. Recombinant E. coli clones were confirmed by colony PCR. Sequence analysis confirmed the presence of the complete gene (2640bp) including start and stop codons. The complete protein sequence consists 879 amino acids. SDS-PAGE and analysis by EXPASy–ProtParam indicated the molecular weight of DNAP-1 as ~92 kDa. Polymerase activity of His-tag purified DNAP-1 was demonstrated by PCR methodology.