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Expression and purification of yeast eRF1 mutant proteins in Escherichia coli
Madayanti F.a, Susilowati P.E.a,b, Aditiawati P.a, Akhmalokaa
a Biochemistry Research Group, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Indonesia
b Departement of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Haluoleo, 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]Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3, which interact to form a heterodimer that mediates termination at all three stop codons. Detailed mechanism of the interaction between the two proteins, however, was still unclear yet. Previous studies indicated that threonin-295 on the third domain of eRF1, involved in its interaction to eRF3. In other to further characterize the role of threonine at position 295 on its interaction, two SUP45 mutants, namely sup45-T29SA and sup45-T295S, were constructed and expressed in Escherichia coli. The mutations were successfully performed by PCR megaprimer and confirmed by sequence analysis. The mutant genes were over expressed in Escherichia coli BL21 (DE3) under the promoter of T7 using pUKC630 vector. The mutant proteins namely, eRF1-T295A and eRF1-T295S, were expressed over 19% and 18% of total protein, respectively. The proteins were successfully purified by one step purification through Immobilized Metal Affinity Chromatography (IMAC).[/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]eRF1,IMAC purification,Over-expression,sup45 mutants[/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]