Expression and efficient purification of tag-cleaved active recombinant human insulin-like growth factor-II from Escherichia coli

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• 作者：Hongbo Li ; Xiaoyan Hui ; Peng Li ; Aimin Xu…
• 关键词：insulin ; like growth factor ; II ; recombinant protein ; protein expression and purification ; enterokinase ; Escherichia coli
• 刊名：Biotechnology and Bioprocess Engineering
• 出版年：2015
• 出版时间：April 2015
• 年：2015
• 卷：20
• 期：2
• 页码：234-241
• 全文大小：782 KB
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• 作者单位：Hongbo Li (1) (2)
  Xiaoyan Hui (3)
  Peng Li (2)
  Aimin Xu (2) (3)
  Shiwu Li (4)
  Shouguang Jin (5)
  Donghai Wu (2)
  
  1. The Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province, Department of Life Sciences, Huaihua College, Huaihua, 418-008, China
  2. The Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
  3. Department of Medicine, The University of Hong Kong, Hong Kong, China
  4. Department of Pathology, University of Florida, Gainesville, USA
  5. Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, USA
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  
• 出版者：The Korean Society for Biotechnology and Bioengineering
• ISSN：1976-3816

文摘

Insulin-like growth factor-II (IGF2) is a growth factor for the control of cell proliferation and apoptosis. To explore the clinical use of human IGF2, an efficient method for production of a large amount of active recombinant hIGF2 is necessary. Human IGF2 cDNA was cloned into pET32 vector where it is under the control of an IPTGinducible T7 promoter. High level soluble thioredoxin (Trx)-hIGF2 fusion protein was produced at room temperature following IPTG induction, amounting up to 20% of the total soluble bacterial proteins. The recombinant Trx-hIGF2 fusion protein was purified to an approximate 95% purity using Ni+-NTA affinity chromatography with an overall yield of 120 mg protein per liter of bacterial culture. After cleavage of the Trx fusion fragment by recombinant enterokinase, the tag-free recombinant hIGF2 protein (rhIGF2) was purified by passage through the Ni+-NTA affinity column again. Biological activity of the purified hIGF2 was determined by its ability to support NIH/3T3 cells proliferation and to activate AKT signaling pathways. Our results demonstrate that tag-free active rhIGF2 can easily be obtained for various applications from E. coli using the procedure described in this report.