Expression and purification of recombinant human neuritin from Pichia pastoris and a partial analysis of its neurobiological activity in vitro

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• 作者：Yunhua Zhang ; Shujun Zhang ; Lingling Xian…
• 关键词：Dorsal root ganglion (DRG) ; Neuritin ; PC12 cells ; Pichia pastoris ; Purification
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2015
• 出版时间：October 2015
• 年：2015
• 卷：99
• 期：19
• 页码：8035-8043
• 全文大小：2,137 KB
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• 作者单位：Yunhua Zhang (1)
  Shujun Zhang (1)
  Lingling Xian (1)
  Juan Tang (1)
  Jingling Zhu (1)
  Lijuan Cui (1)
  Shanman Li (2)
  Lei Yang (3)
  Jin Huang (1)
  
  1. The Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Department of Biochemistry, School of Medicine, Shihezi University, Shihezi, 832002, Xinjiang, China
  2. Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi, 832003, Xinjiang, China
  3. School of Medicine, Hangzhou Normal University, Hangzhou, 310036, Zhejiang, China
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

Neuritin (also known as candidate plasticity gene 15 (cpg15)) is a neurotrophic factor that was recently discovered in a screen aimed at identifying genes involved in activity-dependent synaptic plasticity. Neuritin plays multiple roles in both neural development (Chen et al. Zhonghua Yan Ke Za Zhi 46:978-83 2010; Corriveau et al. J Neurosci 19:7999-8008 1999; Lee and Nedivi J Neurosci 22:1807-1815 2002) and synaptic plasticity (Fujino et al. Gene Dev 25:2674-2685 2011; Leslie and Nedivi Prog 14 Neurobiol 94:223-237 2011; Loebrich and Nedivi Physiol Rev 89:1079 2009). In this study, to produce bioactive, soluble recombinant human neuritin protein, a portion of NRN1 was cloned into the Pichia pastoris expression vector pPIC9K. The recombinant vector was then transformed into the methylotrophic yeast strain P. pastoris GS115, and a shaking flask method and His-tag purification strategy were utilized to express and purify neuritin protein. The resulting protein had a molecular mass of approximately 11 kDa, and subsequent functional analyses indicated that the purified neuritin promoted neurite outgrowth from embryonic chicken dorsal root ganglions, while also prolonging the survival of these ganglions, and from PC12 cells. These findings suggest that neuritin was expressed effectively in vitro and that this protein may play a role in stimulating neurite outgrowth of both dorsal root ganglions and PC12 cells. This study provides a novel strategy for the large-scale production of bioactive neuritin, which will enable further study of the biological function of this protein. Keywords Dorsal root ganglion (DRG) Neuritin PC12 cells Pichia pastoris Purification