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Construction of synthetic open reading frame encoding human interferon alpha 2b for high expression in Escherichia coli and characterization of its gene product
Retnoningrum D.S.a, Ningrum R.A.a, Kurniawan Y.N.a, Indrayati A.a, Rachmawati H.a
a School of Pharmacy, 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]The aim of this research was to obtain recombinant human interferon alpha 2b (rhIFNα2b) from a synthetic open reading frame (ORF) overexpressed in Escherichia coli. For gene assembly, oligonucleotides were designed by Thermodynamically Balanced Inside Out (TBIO) method using the published synthetic codon optimized hIFNα2b ORF for high expression in E. coli. The synthetic ORF was assembled by a two-step Polymerase Chain Reaction (PCR) and cloned into a pGEM-T vector. The two-step PCR resulted in a DNA band of 522 base pairs (bp) corresponding to the size of hIFNα2b ORF. Fifteen recombinant pGEM-Ts were obtained and the sequencing results showed that the ORFs contained one to ten mutations with an error rate of 8.3 per kilo base. An ORF carrying one mutation was cloned into a pET32b vector and site-directed mutagenesis was performed to correct the mutation. The hIFNα2b ORF was overexpressed as a thioredoxin-his-tag fusion protein in E. coli BL21. The rhIFNα2b fusion protein was isolated from inclusion bodies (IB), renatured, and purified using Nickel columns, and all steps were monitored by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). A rhIFNα2b fusion protein of 37 kDa in size was produced in high expression levels relative to total protein, renatured and purified from IB with a yield of 3.46 mg/l without any further optimization. The purified rhIFNα2b was confirmed by peptide analysis with nano-LC-MS/MS2 mass spectrometry. Our current research demonstrates for the first time that by using the TBIO method a synthetic ORF encoding hIFNα2b gene can be expressed at high levels in E. coli. © 2009 Elsevier B.V. All rights reserved.[/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]Base pairs,Codon optimization,DNA bands,E. coli,Error rate,Expression in Escherichia coli,Expression levels,Fusion proteins,Gene assembly,Gene products,His-tag,Inclusion bodies,Nano-LC,Nickel column,Open reading frame,Peptide analysis,Polymerase chain reaction,Recombinant human interferon,SDS-PAGE,Site directed mutagenesis,Sodium dodecyl sulphate,Thioredoxins,Total protein[/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]Codon optimization,Escherichia coli,High expression,Human interferon α2b,Inclusion bodies,Synthetic ORF[/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 financially supported by grant from Ministry of Research and Technology, Indonesia (number: 53/RT/Insentif/PPK/II/2008 ). The authors would like to thank Dr. Oliver Valerius of Institute for Microbiology and Genetics, Georg-August University of Gottingen, Germany for fruitful discussion results and for his courtesy to be involved in LC–MS/MS 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=”DOI” size=”size-sm” text_align=”text-left”][vc_column_text]https://doi.org/10.1016/j.jbiotec.2009.11.008[/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]