Preparation and characterization of a potent, long-lasting recombinant human serum albumin-interferon-α2b fusion protein expressed in Pichia pastoris
| Autor: | Tong-Ying Wang, Yan-Shan Huang, Lin-Fu Zhou, Yang Zhiyu, Zhi Chen, Li Zhou, Jian-Bing Wu |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Chemistry
Pharmaceutical Recombinant Fusion Proteins Molecular Sequence Data Serum albumin Pharmaceutical Science Interferon alpha-2 Protein Sorting Signals Antiviral Agents Pichia law.invention Pichia pastoris Affinity chromatography law medicine Humans Amino Acid Sequence Serum Albumin Chromatography biology Interferon-alpha Biological activity General Medicine Chromatography Ion Exchange Human serum albumin biology.organism_classification Fusion protein Molecular biology Recombinant Proteins Molecular Weight Biochemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein Recombinant DNA Isoelectric Focusing Biotechnology medicine.drug |
| Zdroj: | European Journal of Pharmaceutics and Biopharmaceutics. 67:301-308 |
| ISSN: | 0939-6411 |
| DOI: | 10.1016/j.ejpb.2007.02.015 |
| Popis: | A long-lasting recombinant human serum albumin-interferon-alpha2b fusion protein (rHSA/IFNalpha2b) was prepared and its structure and biological activities were studied. rHSA/IFNalpha2b was expressed in methylotrophic yeast Pichia pastoris with HSA's natural signal peptide and purified by dye affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography and Sephadex G25. Purity of the prepared rHSA/IFNalpha2b was greater than 97% analyzed by non-reduced SDS-PAGE and RP-HPLC. Structure and biological activities of the prepared rHSA/IFNalpha2b were characterized by physical, chemical and biological methods. Its pI was 5.3 and showed a single band on IEF gel. Molecular weight determined by MALDI-TOF was 86004.3+/-29.2. Amino-terminal and carboxyl-terminal amino acid sequences were identical to predicted sequence. Its specific activity in vitro was 6.3+/-0.8x10(5) IU/mg fusion protein, retaining about 1.4% of that of unmodified rIFNalpha on a molar basis. After administered in monkeys, significant increases of 2',5'-oligoadenylate synthetase activity relative to IFN-alpha were maintained for 14 days in serum and the rHSA/IFNalpha2b showed more potent biological activity than IFN-alpha on a molar basis. Therefore, markedly improved in vivo biological activity of rHSA/IFNalpha2b could exhibit more potent antiviral activity than IFNalpha2b in future clinical trials. |
| Databáze: | OpenAIRE |
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