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Construction, expression and purification of DNA Pol I ITB1 and its deletion mutants
Helwati H.a, Hertadi R.a, Madayanti F.a, Akhmalokaa
a Biochemistry Research Group, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Indonesia
[vc_row][vc_column][vc_row_inner][vc_column_inner][vc_separator css=”.vc_custom_1624529070653{padding-top: 30px !important;padding-bottom: 30px !important;}”][/vc_column_inner][/vc_row_inner][vc_row_inner layout=”boxed”][vc_column_inner width=”3/4″ css=”.vc_custom_1624695412187{border-right-width: 1px !important;border-right-color: #dddddd !important;border-right-style: solid !important;border-radius: 1px !important;}”][vc_empty_space][megatron_heading title=”Abstract” size=”size-sm” text_align=”text-left”][vc_column_text]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.[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Author keywords” size=”size-sm” text_align=”text-left”][vc_column_text][/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Indexed keywords” size=”size-sm” text_align=”text-left”][vc_column_text]Deletion mutant,DNA polymerase I,Heterologous expression,His-tag[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Funding details” size=”size-sm” text_align=”text-left”][vc_column_text][/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”DOI” size=”size-sm” text_align=”text-left”][vc_column_text][/vc_column_text][/vc_column_inner][vc_column_inner width=”1/4″][vc_column_text]Widget Plumx[/vc_column_text][/vc_column_inner][/vc_row_inner][/vc_column][/vc_row][vc_row][vc_column][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][/vc_column][/vc_row]