title: Production of human mutant biologically active hepatocyte growth factor in Chinese hamster ovary cells.
abstract: Hepatocyte growth factor (HGF) is a potent multifunctional cytokine that affects proliferation, migration, and morphogenesis of various cells. HGF is secreted as an inactive single-chain precursor protein and activated by the cleavage of serine proteases to form heterodimers. In our current study, the cleavage site of HGF was blocked by replaced Arg 494 of Glu (R494E) that resulted in the single-chain HGF (R494E) unable to be cleaved by serine proteases. We established Chinese hamster ovary (CHO) cells overexpressing HGF (R494E), the expression of HGF (R494E) achieved 12 mg/L and was similar to a previously reported study. The recombinant protein was then purified from culture medium using a two-step chromatographic procedure that resulted in about a 40% recovery rate. The purified HGF (R494E) was obtained as a single-chain active protein. It concluded that HGF (R494E) exhibited a biologically active protein and the overexpressing CHO cell line supplied sufficient material for future studies. The R494E replacement of the cleavage site would be beneficial to the utility of other similar therapeutic proteins.
authors.affiliation: a Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Xu
authors.affiliation: a Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
authors.forename: Aini
authors.initials: A
authors.lastname: Wan
authors.affiliation: a Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
authors.forename: Lin
authors.initials: L
authors.lastname: Peng
authors.affiliation: a Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Chen
authors.affiliation: b Jiangsu Institute of Hematology , The First Affiliated Hospital of Soochow University , Suzhou , China.
authors.forename: Yang
authors.initials: Y
authors.lastname: He
authors.affiliation: c Cyrus Tang Hematology Center and Ministry of Education Engineering Center of Hematological Disease , Soochow University , Suzhou , China.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Yang
authors.affiliation: a Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
authors.forename: Jian
authors.initials: J
authors.lastname: Jin
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001709:Biotechnology; D016466:CHO Cells; D016923:Cell Death; D002460:Cell Line; D024042:Collagen Type I / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004285:Dogs; D017228:Hepatocyte Growth Factor / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: D024042:Collagen Type I; C066855:HGF protein, human; D011994:Recombinant Proteins; D017228:Hepatocyte Growth Factor
keywords: Chinese hamster ovary; heterodimers; recombinant HGF; single chain