Current location: JICS Archive > Vol. 6 > No. 4 > Articles : 22
Molecular Cloning, Expression and Sequence Analysis of DNA Polymerase I from an Iranian Thermophilic Bacterium, Bacillus sp. G (2006)
E. Farashahi Yazda, M. Sadeghizadeha, š* S. Hosseinkhanib, M. Khalaj-Kondoria and R. Emamzadehb
aDepartment of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Islamic Republic of Iran
bDepartment of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Islamic Republic of Iran
Thermostable DNA polymerases are widely used in DNA amplification reactions such as the Polymerase Chain Reaction (PCR), requiring the activity of the enzymes at high temperatures. The aim of the present study was to assess the potential biotechnological capabilities of Iranian thermostable DNA polymerases. To this end, we cloned the gene encoding a DNA polymerase from a novel thermophilic eubacterium, Bacillus sp. G (2006). Phylogentic analysis of this gene revealed that the new isolate belongs to the genera Bacillus. Sequence analysis of the fragment produced by degenerate primers also showed that it consists of 2,631 bp encoding an 876 amino acid protein, and subsequent amino acid sequence analysis of this DNA polymerase showed that it belongs to family A-type DNA polymerases. The expression vector pET28a (+) was chosen for expression of the gene fragment in the mesophilic host bacterium E. coli BL21. This expression vector has some advantages such as attachment of a Poly-His tag to the N-terminus of the protein for the ease of purification and a powerful promoter of lac-Z induced by IPTG. The band corresponding to the protein product was observed in the molecular weight range of about 100KDa on the SDS-PAGE gel after heat and Ni+2-NTA column chromatography. Using the dot blot technique, the polymerase activity of the enzyme was qualitatively confirmed at 70 °C. Therefore, it is suggested that optimizations of this activity could make this enzyme appropriate for PCR processes in future.
Keywords: Cloning, Expression, Thermophile, DNA polymerase I
