Journal of Pure and Applied MicrobiologyVol. 3 No. 2

Construction, Expression and Purification of DNA Pol I ITB1 and its Deletion Mutants

Hira Helwati, Rukman Hertadi, Fida Madayanti and Akhmaloka*

Biochemistry Research Group, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jln Ganesha 10, Bandung. Indonesia.

Received on 18 June 2009 and accepted on 27 July 2009

 

ABSTRACT

A gene encoding DNA polymerase I, namely DNA Pol I ITB1, was cloned from Geobacillus thermoleovorans. Heterologous expression of DNA Pol I ITB1 in Escherichia coli was carried out under expression vector pET30a(+). The vector contains his-tagged in the upstream of the gene, thus the expressed protein carried poly his on the N-terminal. Recombinant plasmid containing DNA Pol I ITB1 gene was constructed by inserting NcoI BamHI gene fragment of pITB8 containing the whole coding region of DNA PolI ITB1 into pET30a(+) resulting pITB20 plasmid. The deletion mutants of the genes were constructed through in frame deletion of the gene by using EcoRI, and XhoI restriction enzymes resulting pITB21, and pITB22 plasmids respectively. The expression of the wild type and the deletion mutants were carried out in Escherichia coli BL21-DE3. High expression levels of the genes were shown on SDS-PAGE. The proteins were purified by immobilized metal-ion affinity chromatography (IMAC) using Ni-NTA resin and single band protein products were shown on the gel following SDS PAGE analysis.

Keywords : DNA Polymerase I, Deletion mutant, Heterologous expression, His-tag.