title: Inhibition of the ubiquitin ligase activity improves the production of biologically active fusion protein HSA-HGF in Chinese hamster ovary cells.
abstract: Hepatocyte growth factor (HGF) is a potent multi-functional protein that stimulates proliferation, survival, motility, scattering and differentiation during growth and development, and has been considered to be a potential therapeutic agent for the treatment of a number of intractable diseases. The aim of this study was to enhance the expression of recombinant fusion protein HSA-HGF (R494E) in CHO cells by inhibiting the intracellular ubiquitin ligase activity. The high stable expression sub-clones with different signal peptides were selected by western blot (WB) analysis and used for suspension culture. We found that the expression of fusion protein HSA-HGF (R494E) on day 3 achieved 50 mg/L during the 8 day culture process, a large number of fusion proteins were intracellular degradated by ubiquitination pathway during day 4 to day 8. Furthermore, ubiquitin ligase inhibitor, thalidomide, was added in culture process, and resulted in efficient and stable secretion of HSA-HGF (R494E) in CHO cells. According to biological activity assays, HSA-HGF (R494E) possessed various biological activities similar to native HGF. In conclusion, innhibition of intracellular ubiquitin ligase activity was successfully improve the expression of biologically active fusion protein HSA-HGF (R494E) in CHO cells. Our data may be beneficial to enhance the production of other therapeutic proteins in fed-batch culture.
authors.affiliation: a The Key Laboratory of Industrial Biotechnology, Ministry of Education , School of Biotechnology, Jiangnan University , Wuxi , China.|b Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Xu
authors.affiliation: a The Key Laboratory of Industrial Biotechnology, Ministry of Education , School of Biotechnology, Jiangnan University , Wuxi , China.|b Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
authors.forename: Aini
authors.initials: A
authors.lastname: Wan
authors.affiliation: b Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: a The Key Laboratory of Industrial Biotechnology, Ministry of Education , School of Biotechnology, Jiangnan University , Wuxi , China.
authors.forename: Lin
authors.initials: L
authors.lastname: Peng
authors.affiliation: b Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Chen
authors.affiliation: c Jiangsu Institute of Hematology , the First Affiliated Hospital of Soochow University , Suzhou , China.
authors.forename: Yang
authors.initials: Y
authors.lastname: He
authors.affiliation: d 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: b Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
authors.forename: Jian
authors.initials: J
authors.lastname: Jin
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D015536:Down-Regulation; D004789:Enzyme Activation; D017228:Hepatocyte Growth Factor / Q000096:biosynthesis* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012709:Serum Albumin / Q000096:biosynthesis* / Q000235:genetics*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D012709:Serum Albumin; D017228:Hepatocyte Growth Factor; D044767:Ubiquitin-Protein Ligases
keywords: CHO; HSA-HGF; biologically active; intracellular degradation