title: Binding and biologic characterization of recombinant human serum albumin-eTGFBR2 fusion protein expressed in CHO cells.
abstract: Transforming growth factor-β1 (TGF-β1) signaling is involved in cell metabolism, growth, differentiation, carcinoma invasion and fibrosis development, which suggests TGF-β1 can be treated as a therapeutic target extensively. Because TGF-β1 receptor type α(TGFBR2) is the directed and essential mediator for TGF-β1 signals, the extracellular domain of TGFBR2 (eTGFBR2), binding partner for TGF-β1, has been produced in a series of expression systems to inhibit TGF-β1 signaling. However, eTGFBR2 is unstable with a short half-life predominantly because of enzymatic degradation and kidney clearance. In this study, a fusion protein consisting of human eTGFBR2 fused at the C-terminal of human serum albumin (HSA) was stably and highly expressed in Chinese Hamster Ovary (CHO) cells. The high and stable expression sub-clones with Ig kappa signal peptide were selected by Western blot analysis and used for suspension culture. After fed-batch culture over 8 d, the expression level of HSA-eTGFBR2 reached 180 mg/L. The fusion protein was then purified from culture medium using a 2-step chromatographic procedure that resulted in 39% recovery rate. The TGF-β1 binding assay revealed that HSA-eTGFBR2 could bind to TGF-β1 with the affinity constant (KDof 1.42 × 10-8M) as determined by the ForteBio Octet System. In addition, our data suggested that HSA-eTGFBR2 exhibited a TGF-β1 neutralizing activity and maintained a long-term activity more than eTGFBR2. It concluded that the overexpressing CHO cell line supplied sufficient recombinant human HSA-eTGFBR2 for further research and other applications.
authors.affiliation: a The Key Laboratory of Industrial Biotechnology , Ministry of Education, School of Biotechnology, 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: Yana
authors.initials: Y
authors.lastname: Miao
authors.affiliation: a The Key Laboratory of Industrial Biotechnology , Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi , China.
authors.forename: Lin
authors.identifier: http://orcid.org/0000-0002-5708-4116
authors.initials: L
authors.lastname: Peng
authors.affiliation: b Laboratory of Molecular Pharmacology , School of Pharmaceutical Sciences, Jiangnan University , Wuxi , China.
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Cai
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
authors.affiliation: a The Key Laboratory of Industrial Biotechnology , Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi , China.
authors.forename: Huazhong
authors.initials: H
authors.lastname: Li
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D003412:Cricetulus; D024861:Genetic Enhancement / Q000379:methods; D006801:Humans; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000737:chemistry*; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000096:biosynthesis* / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D012709:Serum Albumin / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics
chemical_list: D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II
keywords: Antagonist; Chinese hamster ovary cells; TGF-β1; eTGFBR2; human serum albumin