title: Efficient production of sTNFRII-gAD fusion protein in large quantity by use of the modified CHO-S cell expression system.
abstract: TNFα is one of the initial and important mediators to activate downstream signaling pathways by binding to trimerized TNFα receptors (TNFR), and thus is an ideal drug target for cancer therapy. Taking advantage of intrinsic homotimerization of the globular domain of adiponectin (gAD), we have developed a novel TNFα antagonist, the trimerized fusion protein named sTNFRII-gAD. However, our previously-used CHO expression system yielded less than 10 mg/L of sTNFRII-gAD. To produce large quantities of sTNFRII-gAD efficiently, we used a modified CHO-S cell expression system, which is based on a pMH3 vector with non-coding GC-rich DNA fragments for high-level gene expression. We obtained stable clones that produced 75 mg/L of sTNFRII-gAD in the 96-well plate, adapted the clones to 40 ml suspension serum-free batch culture, then optimized the culturing conditions to scale up the fed-batch culture in a 3 L shake-flask and finally in a 5 L AP30 bioreactor. We achieved a final yield of 52 mg/L of sTNFRII-gAD. The trimerized sTNFRII-gAD exhibited the higher affinity to TNFα with a dissociation constant (Kd) of 5.63 nM than the dimerized sTNFRII-Fc with a Kd of 13.4 nM, and further displayed the higher TNFα-neutralizing activity than sTNFRII-Fc (p<0.05) in a L929 cytotoxicity assay. Therefore, the strategy employed in this study may provide an efficient avenue for large-scale production of other recombinant proteins by use of the modified CHO-S cell expression system.
authors.affiliation: Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, School of Laboratory Medicine & Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Qinzhen
authors.initials: Q
authors.lastname: Cai
authors.affiliation: Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, School of Laboratory Medicine & Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Ai
authors.initials: A
authors.lastname: Zhao
authors.affiliation: Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, School of Laboratory Medicine & Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.lastname: Yuting Yin
authors.affiliation: Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, School of Laboratory Medicine & Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Lisha
authors.initials: L
authors.lastname: Ma
authors.affiliation: Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, School of Laboratory Medicine & Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Zhenzhen
authors.initials: Z
authors.lastname: Jiao
authors.affiliation: Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, School of Laboratory Medicine & Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Huilin
authors.initials: H
authors.lastname: Zhi
authors.affiliation: Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, School of Laboratory Medicine & Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Shouhua
authors.initials: S
authors.lastname: Lai
authors.affiliation: Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, School of Laboratory Medicine & Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Sha
authors.initials: S
authors.lastname: Cheng
authors.affiliation: Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, School of Laboratory Medicine & Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Yang
authors.affiliation: Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, School of Laboratory Medicine & Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Yinxiang
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Departments of Immunology and Molecular Genetics, University of Toronto, Mount Sinai Hospital, Samuel Lunenfeld Research Institute and Toronto General Research Institutes, Toronto, Ontario, Canada.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Siminovitch
authors.affiliation: Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, School of Laboratory Medicine & Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Jimin
authors.initials: J
authors.lastname: Gao
mesh_terms: D052242:Adiponectin / Q000096:biosynthesis*; D000818:Animals; D001681:Biological Assay; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry; D004262:DNA Restriction Enzymes; D020869:Gene Expression Profiling; D006801:Humans; D010957:Plasmids; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors
chemical_list: D052242:Adiponectin; D003470:Culture Media; D047889:Receptors, Tumor Necrosis Factor, Type II; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D004262:DNA Restriction Enzymes