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Expression of rMnSODSeq regulated by the auto-inducible dps promoter in Escherichia coli
Anindyajatia, Riani C.a, Karya A.R.O.a, Retnoningrum D.S.a
a Laboratory of Pharmaceutical Biotechnology, Pharmaceutics Department, School of Pharmacy Institut Teknologi Bandung, Bandung, 40132, 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]© 2019 Elfos Scientiae. All rights reserved.Recombinant protein production has been favored in Escherichia coli by using the T7 promoter, strongly regulated by IPTG, as a strategy for high transcriptional efficiency and to regulate protein overexpression. Unfortunately, IPTG is expensive and could be toxic to cells. In this setting, auto-inducible promoters emerged for recombinant protein production, as the dps promoter, which is growth phase-dependent and active in stationary phase. Previously, the plasmid pCAD-sod construct was generated, expressing the rMnSODSeq gene expression cassette under the dps promoter, and rMnSODSeq protein was expressed in E. coli. In this work, the rMnSODSeq overproduction using pCAD-sod under the regulation of dps promoter is characterized in E. coli TOP10. The influence of the incubation time and growth medium variables was analyzed. Protein yield was determined by SDS-PAGE assisted with ImageJ software, the protein was purified using a Ni2+-NTA column, and its activity was confirmed using zymography. Lastly, plasmid stability in E. coli TOP10 grown in both Luria-Bertani (LB) and Terrific Broth (TB) media were also assessed. Optimized overproduction was attained in 200 mL culture of both LB and TB at 37 °C and 150 rpm for 24 h. Overproduction in TB provided higher cell densities, while SOD percentage of total protein in LB reached the highest values (48.70 ± 1.15 %). Protein purification yielded rMnSODSeq with electrophoretic purity above 90 % and zymography analysis confirmed that the protein showed dismutation activity. The use of dps auto-inducible promoter in pCAD-sod plasmid for rMnSODSeq overproduction in E. coli system is promising for further upscaling.[/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]Auto-inducible dps promoter,Escherichia coli,PCAD-sod,RMnSODSeq[/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][{‘$’: ‘This work was funded by Program Penelitian, Pengabdian Masyarakat, dan Inovasi (P3MI) Kelompok Keahlian Grant from Institut Teknologi Bandung, 2017.’}, {‘$’: ‘This work was funded by Program Penelitian, Pen-gabdian Masyarakat, dan Inovasi (P3MI) Kelompok Keahlian Grant from Institut Teknologi Bandung, 2017.’}][/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]