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All Journal HAYATI Journal of Biosciences ANNALES BOGORIENSES Narra J Makara Journal of Science Annales Bogorienses
Andri Wardiana, Andri
Research Center for Biotechnology, Indonesian Institute of Sciences Jl. Raya Bogor KM 46, Cibinong, Bogor, 16911, Indonesia
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Earth & Planetary Sciences Health Professions Immunology & microbiology Medicine & Pharmacology Public Health

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Expression of No Affinity Tagged Recombinant Human Interferon Alpha-2a in Methylotrophic Yeast Pichia pastoris Herawati, Neng; Wardiana, Andri; Ningrum, Ratih Asmana
Annales Bogorienses Vol. 19 No. 2 (2015): Annales Bogorienses
Publisher : BRIN

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Abstract

Recombinant human interferon alpha-2a (rhIFN-2a) has been widely used for clinical therapy as antiviral, anticancer as well as immunomodulator. In this study, the open reading frame (ORF) encoding synthetic hIFN-2a was constructed to be in framed with N-terminal alpha factor secretion system in methylotrophic yeast Pichia pastoris. This research aimed to construct, express and analyse the non-affinity tagged recombinant human interferon alpha-2a in the methylotrophic yeast P. pastoris. We used pPICZB plasmid for cloning and expression vector. The confirmed recombinant plasmid containing the correct DNA sequence of hIFN-2a was linearized by SacI restriction enzyme, then transformed into P. pastoris genome using electroporation. We screened two multi-copy recombinants in YPDS plates containing Zeocin™. Buffered complex medium containing 0.5 % methanol (BMMY) was used for protein expression for 48 hours in the culture condition. The recombinant protein was purified by blue sepharose affinity chromatography. Analyses of hIFN-2a protein by SDS-PAGE and Western blot confirmed that protein band in which was observed around 19.2 kDa, was recombinant hIFN-2a. The quantification of purified rhIFN-2a using colorimetric binichoninic assay (BCA) informed that the yield was 44 mg/L culture (OD600= 2-3). 
Co-Authors Adi Santoso Adzdzakiy, Muhammad M. Agustiyani, Dian F. Agustiyanti, Dian Fitria Agustus Sani Nugroho & Ema Rahmawati Heryaman, Agustus Sani Nugroho & Alamanda, Cut NC. Asyifa, Isnaini Z. Azzania Fibriani Desriani Desriani Dharmayanthi, Anik B. Ernawati A. Giri Rachman Ferry Iskandar Hariyatun, Hariyatun Husnaa, Ulfatul Husnaa, Ulfatul Iryanto, Syam B. Kusumawati, Arizah Kusumawati, Arizah Marissa Angelina Nasrulloh, Mukh F. Neng Herawati, Neng Ningrum, Ratih A. Nugroho, Herjuno A. Permatasari, Fitri Aulia Popi Hadi Wisnuwardhani, Popi Hadi Prasetyoputri, Anggia Rachman, Rifky W. RATIH ASMANA NINGRUM Ristandi, Ryan B. Robiani, Rini Saputra, Sugiyono Sativa, Alvira R. Stephanie, Rebecca Taharuddin, Audrey Angelina Putri Wijaya, Sri Kartika Wijaya, Sri Kartika Wisnuwardhani, Popi H. Wulandari, Ari S. Yamahoki, Nicholas Yana Rubiana, Yana Yana Rubiyana, Yana Yanthi, Nova D.