Efficient Production of sTNFRII-gAD Fusion Protein in Large Quantity by Use of the Modified CHO-S Cell Expression System

doi:10.1371/journal.pone.0111229

科研成果详情

题名	Efficient Production of sTNFRII-gAD Fusion Protein in Large Quantity by Use of the Modified CHO-S Cell Expression System
作者	Cai, Qinzhen1; Zhao, Ai1; Yin, Yuting1; Ma, Lisha 1; Jiao, Zhenzhen 1; Zhi, Huilin 1; Lai, Shouhua1; Cheng, Sha 1; Yang, Hongmei1; Lu, Yinxiang 1; Siminovitch, Katherine A.2; Gao, Jimin1
发表日期	2014-10-23
发表期刊	PLOS ONE 影响因子和分区
语种	英语
原始文献类型	Article
其他关键词	HAMSTER OVARY CELLS ; LOW CULTURE TEMPERATURE ; BATCH CULTURES ; RECEPTORS ; AGENTS ; METABOLISM ; ADIPOSE ; GAMMA
摘要	TNF alpha is one of the initial and important mediators to activate downstream signaling pathways by binding to trimerized TNF alpha 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 TNF alpha 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 TNF alpha 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 TNF alpha-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.
资助项目	National Major Specific Project for Innovation of New Pharmaceuticals [2009ZX09103-649]; Chinese National 863 plan [2012AA02A407]; Scientific Research Fund of Ministry of Public Health [201231029]; Zhejiang Provincial Major Research Program [2010C13007]; Key Science and Technology Innovation Team of Zhejiang Province; Natural Science Foundation of Zhejiang ProvinceNatural Science Foundation of Zhejiang Province [Y2110580, LY12C07001, LY13H100003, LZ14H260001]; Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents; Science & Technology Innovation Program for College/University Students in Zhejiang Province [2012R413039, 2012R413041]; Wenzhou Municipal Research Program [Y20100183, Y20100274]
出版者	PUBLIC LIBRARY SCIENCE
出版地	SAN FRANCISCO
ISSN	1932-6203
卷号	9 期号:10 页码:e111229.
DOI	10.1371/journal.pone.0111229
页数	9
WOS类目	Multidisciplinary Sciences
WOS研究方向	Science & Technology - Other Topics
WOS记录号	WOS:000343662800095
收录类别	SCIE ; PUBMED ; SCOPUS
URL	查看原文
PubMed ID	25340707
PMC记录号	PMC4207793
SCOPUSEID	2-s2.0-84908565970
通讯作者地址	[Cai, Qinzhen]Zhejiang Provincial Key Laboratory for Technology and Application of Model Organisms, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University,Wenzhou, Zhejiang,China
Scopus学科分类	Multidisciplinary
引用统计	被引频次：2[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	https://kms.wmu.edu.cn/handle/3ETUA0LF/10613
专题	检验医学院（生命科学学院、生物学实验教学中心）_模式生物技术与应用重点实验室
通讯作者	Cai, Qinzhen
作者单位	1.Zhejiang Provincial Key Laboratory for Technology and Application of Model Organisms, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University,Wenzhou, Zhejiang,China; 2.Departments of Immunology and Molecular Genetics, University of Toronto, Mount Sinai Hospital,Toronto,Canada
第一作者单位	模式生物技术与应用重点实验室; 检验医学院（生命科学学院、生物学实验教学中心）
通讯作者单位	模式生物技术与应用重点实验室; 检验医学院（生命科学学院、生物学实验教学中心）
第一作者的第一单位	模式生物技术与应用重点实验室
推荐引用方式 GB/T 7714	Cai, Qinzhen,Zhao, Ai,Yin, Yuting,et al. Efficient Production of sTNFRII-gAD Fusion Protein in Large Quantity by Use of the Modified CHO-S Cell Expression System[J]. PLOS ONE,2014,9(10):e111229..
APA	Cai, Qinzhen., Zhao, Ai., Yin, Yuting., Ma, Lisha., Jiao, Zhenzhen., ... & Gao, Jimin. (2014). Efficient Production of sTNFRII-gAD Fusion Protein in Large Quantity by Use of the Modified CHO-S Cell Expression System. PLOS ONE, 9(10), e111229..
MLA	Cai, Qinzhen,et al."Efficient Production of sTNFRII-gAD Fusion Protein in Large Quantity by Use of the Modified CHO-S Cell Expression System".PLOS ONE 9.10(2014):e111229..