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Andri Wardiana
Research Center for Biotechnology-LIPI
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Immunology & microbiology Medicine & Pharmacology

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Expression of No Affinity Tagged Recombinant Human Interferon Alpha-2a in Methilotropic Yeast Pichia pastoris Neng Herawati; Andri Wardiana; Ratih Asmana Ningrum
ANNALES BOGORIENSES Vol 19, No 2 (2015): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ann.bogor.2015.v19.n2.57-62

Abstract

Recombinant human interferon alpha-2a (rhIFNα-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 hIFNα-2a was constructed to be in framed with N-terminal alpha factor secretion system in methylotropic yeast Pichia pastoris. This research aimed to construct, express and analyse the non-affinity tagged recombinant human interferon alpha-2a in the methilotropic yeast P. pastoris. We used pPICZαB plasmid for cloning and expression vector. The confirmed recombinant plasmid containing the correct DNA sequence of hIFNα-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 hIFNα-2a protein by SDS-PAGE and Western blot confirmed that protein band in which was observed around 19.2 kDa, was recombinant hIFNα-2a. The quantification of purified rhIFNα-2a using colorimetric binichoninic assay (BCA) informed that the yield was 44 mg/L culture (OD600= 2-3).
Co-Authors Neng Herawati Ratih Asmana Ningrum