Overproduction, characterization and preliminary antiproliferative activity determination of non-tagged recombinant human interferon alpha-2a produced in Pichia pastoris
| Autor: | Andri Wardiana, Ratih Asmana Ningrum, Neng Herawati |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
biology Chemistry Biological activity biology.organism_classification law.invention Pichia pastoris 03 medical and health sciences Open reading frame 030104 developmental biology 0302 clinical medicine lcsh:Biology (General) Biochemistry Affinity chromatography Shuttle vector Cell culture law 030220 oncology & carcinogenesis Recombinant DNA lcsh:QH301-705.5 Peptide sequence |
| Zdroj: | Nusantara Bioscience (2017) |
| ISSN: | 2087-3956 2087-3948 |
| Popis: | Herawati N, Wardiana A, Ningrum RA. 2017. Overproduction, characterization and antiproliferative activity determination of non-tagged recombinant human interferon alpha-2a produced in Pichia pastoris. Nusantara Bioscience 9: 97-101. Recombinant human interferon alpha-2a (rhIFNα-2a) has been widely used for antiviral, anticancer as well as immunomodulatory clinical therapy. In our previous research, we constructed the Open Reading Frame (ORF) encoding synthetic rhIFNα-2a to be in framed with N-terminal alpha factor secretion system in pPICZαB shuttle vector. This research covered overproduction, characterization of the protein and preliminary determination of biological potency assay by using the estrogen positive cell line MCF-7. To overproduce the protein, cultivation was performed in Buffered complex medium containing glycerol (BMGY) for 24 h and production was performed in Buffered complex medium containing methanol (BMMY) during 48 hours at 30°C. The recombinant protein was purified by affinity chromatography using blue sepharose resin. Analysis of amino acid sequence by using MS/MS2 mass spectrometry covered 21% of the total amino acid residues. To determine the initial biological activity, the effect of rhIFNα-2a on MCF-7 breast cancer cell line was studied in vitro. The anti-proliferative activity of hIFNα-2a was performed by 3-[4.5-dimethyltiazol-2il]-2.5 diphenylltetrazolium bromide (MTT) assay. The result showed that the protein is able to inhibit MCF-7 proliferation and the reduction of cell growth was dose-dependently. To monitor the protein production, we performed stability expression assay as well. The result informed that the expression of ORF still stable until 60th generation |
| Databáze: | OpenAIRE |
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