title: Development and characterization of a novel fusion protein of a mutated granulocyte colony-stimulating factor and human serum albumin in Pichia pastoris.
abstract: The purpose of the present work was to develop a novel, long-acting and potent human serum albumin/granulocyte colony stimulating factor (HSA/G-CSF) therapeutic fusion protein. The novel fusion protein, called HMG, was constructed by genetically fusing mutated human derived G-CSF (mG-CSF) to the C-terminal of HSA and then prepared in Pichia pastoris. The molecular mass of HMG was about 85 kDa and the isoelectric point was 5.3. Circular dichroism spectroscopy suggested that mG-CSF retained nearly all of its native secondary structure, regardless of fusion. The binding capabilities of mG-CSF moiety to G-CSF receptor and HSA moiety to warfarin showed very little change after fusing. The bioactivity of HMG (11.0×10(6) IU/mg) was more than twice that of rHSA/G-CSF (4.6×10(6) IU/mg). A mutation was made at the 718th amino acid of HMG, substituting Ala for Thr, to investigate the glycosylation of HMG expressed in P. pastoris. Data indicated that HMG was modified at Thr718, speculatively with the addition of a mannose chain. In conclusion, a novel HSA/G-CSF fusion protein was successfully constructed based on a mutated G-CSF. This protein showed more potent bioactivity than rHSA/G-CSF and thus may be a suitable long-acting G-CSF.
authors.affiliation: Medical Biotechnology Laboratory, Zhejiang University, Hangzhou, China.
authors.forename: Yan-Shan
authors.initials: YS
authors.lastname: Huang
authors.affiliation: Medical Biotechnology Laboratory, Zhejiang University, Hangzhou, China.
authors.forename: Xiao-Fang
authors.initials: XF
authors.lastname: Wen
authors.affiliation: Jiangsu T-Mab Biopharmaceutical Co., Ltd., Jiangsu, China.
authors.forename: Zhi-Yu
authors.initials: ZY
authors.lastname: Yang
authors.affiliation: Jiangsu T-Mab Biopharmaceutical Co., Ltd., Jiangsu, China.
authors.forename: Yi-Liang
authors.initials: YL
authors.lastname: Wu
authors.affiliation: Jiangsu T-Mab Biopharmaceutical Co., Ltd., Jiangsu, China.
authors.forename: You
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Medical Biotechnology Laboratory, Zhejiang University, Hangzhou, China.
authors.forename: Lin-Fu
authors.initials: LF
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000925:Anticoagulants / Q000378:metabolism; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D017433:Protein Structure, Secondary; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000075462:Serum Albumin, Human; D014859:Warfarin / Q000378:metabolism
chemical_list: C422455:ALB protein, human; D000925:Anticoagulants; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D016179:Granulocyte Colony-Stimulating Factor; D014859:Warfarin; D000075462:Serum Albumin, Human