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Subcloning of genes encoding cytochrome P450 monooxygenase into expression vector in Escherichia coli
Wicaksono I.A.a, Lestari T.b, Ulfa E.U.c, Riani C.d, Elfahmi E.d
a Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia
b Department of Biology Pharmacy, STIKes Bakti Tunas Husada, Indonesia
c Department of Biology Pharmacy, Faculty of Pharmacy, Universitas Jember, Indonesia
d Department of Pharmacetics, 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]© 2017 The Authors.Objective: Cytochrome P450 monooxygenase (CYP71AVI) is a key enzyme involved in the artemisinin biosynthesis pathway. In this research, subcloning gene encoding CYP71AVI into pETDUET1 vector in Escherichia coli has been done and then the expression products characterized with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Materials and Methods: Gene construction started with subcloning of cyp71avi gene from pJexpress401_cyp into pETDUET1 through restriction site NdeI and XhoI to get pETDUET1_cyp. Overproduction of CYP71AVI at temperature 37°C has conducted by isopropyl-β-D-thiogalactopyranoside induction. Results: Confirmation of the recombinant vector pETDUET1_cyp was done by migration, restriction site, and sequencing analysis. The result of pETDUET1_cyp restriction analysis with XhoI restriction enzyme showed one DNA band with experimental size 6585 base pair. The CYP71AVI protein has been produced and characterized with SDS-PAGE method. Based on experimental calculation from SDS-PAGE analysis obtained molecular weight of CYP71AVI band was 57.55 kDa. Conclusion: Construction of gene encoding CYP71AVI into pETDUET1 as the co-expression vector in E. coli has been successfully and confirmed by migration, restriction site, and sequencing analysis. The result of overproduction showed protein bands on SDS-PAGE analysis indicated as CYP71AVI.[/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]Cytochrome P450 monooxygenase,Eshcerichia coli,pETDUET1,pETDUET1_cyp[/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]https://doi.org/10.22159/ajpcr.2017.v10s2.19491[/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]