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 pITB_21, 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.