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ABSTRACT: The plasma half-life of therapeutic proteins is a critical factor in many clinical applications. Therefore, new strategies to prolong plasma half-life of long-acting peptides and protein drugs are in high demand. Here, we designed an artificial gelatin-like protein (GLK) and fused this hydrophilic GLK polymer to granulocyte-colony-stimulating factor (G-CSF) to generate a chimeric GLK/G-CSF fusion protein. The genetically engineered recombinant GLK/G-CSF (rGLK/G-CSF) fusion protein was purified from Pichia pastoris. In vitro studies demonstrated that rGLK/G-CSF possessed an enlarged hydrodynamic radius, improved thermal stability and retained full bioactivity compared to unfused G-CSF. Following a single subcutaneous administration to rats, the rGLK/G-CSF fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating white blood cells than G-CSF. Our findings indicate that fusion with this artificial, hydrophilic, GLK polymer provides many advantages in the construction of a potent hematopoietic factor with extended plasma half-life. This approach could be easily applied to other therapeutic proteins and have important clinical applications.
European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 12/2009; 74(3):435-41. · 3.15 Impact Factor
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ABSTRACT: 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.
European Journal of Pharmaceutics and Biopharmaceutics 10/2007; 67(2):301-8. · 4.27 Impact Factor
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ABSTRACT: To reduce the serum clearance of interferon alpha2b, a chimeric gene encoding an human serum albumin(HSA)--human interferon alpha2b(IFNalpha2b) fusion protein was overexpressed in Pichia pastoris. After fermentation in a 5L bioreactor, the fusion protein, capable of cross-reacting with anti-IFN alpha and anti-HSA antibody, was purified from the culture of the recombinant yeast by ultrafiltration, blue Sepharose affinity, phenyl hydrophobic interaction and Q ion exchange chromatography. Its IFNa2b moiety exhibits antiviral activity similar to that of recombinant human IFNa2b. In Cynomolgus monkeys model, The fusion protein was detectable in plasma, even 336h after a single does of 90 microg/kg injection intravenously or subcutaneously. The elimination phase half-life of the fusion protein was 101h after intravenous injection and 68.2h after subcutaneous injection. Its Subcutaneous bioavailability was 67.9%. The enhanced pharmacokinetics of interferon a2b fused to human serum albumin suggest its promissing application in clinic medicine.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 04/2006; 22(2):173-9.
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ABSTRACT: To reduce the serum clearance of interferon α2b, a chimerical gene encoding a human serum albumin (HSA)-human interferon α2b (IFNα2b) fusion protein was over expressed in Pichia pastoris. After fermenting it in a 5 L bioreactor, the fusion protein, capable of cross-reacting with anti-IFN α and anti-HSA antibody, was purified from the culture of the recombinant yeast by ultra-filtration, Blue Sepharose affinity, phenyl hydrophobic interaction and Q ion exchange chromatography. Its IFNα2b moiety exhibits antiviral activity similar to those of recombinant human IFNα2b. In Cynomolgus monkey model, the fusion protein was detectable in plasma, even 336 h after a single dose of 90 μg/kg was injected intravenously or subcutaneously. The elimination phase half-life of the fusion protein was 101 h after intravenous injection and 68.2 h after subcutaneous injection. Its subcutaneous bioavailability was 67.9 %. The enhanced pharmacokinetics of interferon α2b fused to human serum albumin suggests its promising application in clinical medicine.
Chinese Journal of Biotechnology.
