Preparation and characterization of a potent, long-lasting recombinant human serum albumin-interferon-alpha2b fusion protein expressed in Pichia pastoris.
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.
Huang
Yan-Shan
YS
Institute of Infectious Diseases, Zhejiang University, Hangzhou, China.
Chen
Zhi
Z
Yang
Zhi-Yu
ZY
Wang
Tong-Ying
TY
Zhou
Li
L
Wu
Jian-Bing
JB
Zhou
Lin-Fu
LF
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology; D002626:Chemistry, Pharmaceutical / Q000379:methods; D002845:Chromatography; D002852:Chromatography, Ion Exchange / Q000379:methods; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry*; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000378:metabolism*; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D011994:Recombinant Proteins; D012709:Serum Albumin / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000998:Antiviral Agents; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012709:Serum Albumin