title: Expression and purification of recombinant human neuritin from Pichia pastoris and a partial analysis of its neurobiological activity in vitro.
abstract: 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 46:978-983 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.
authors.affiliation: The Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Department of Biochemistry, School of Medicine, Shihezi University, Shihezi, 832002, Xinjiang, China.
authors.forename: Yunhua
authors.initials: Y
authors.lastname: Zhang
authors.forename: Shujun
authors.initials: S
authors.lastname: Zhang
authors.forename: Lingling
authors.initials: L
authors.lastname: Xian
authors.forename: Juan
authors.initials: J
authors.lastname: Tang
authors.forename: Jingling
authors.initials: J
authors.lastname: Zhu
authors.forename: Lijuan
authors.initials: L
authors.lastname: Cui
authors.forename: Shanman
authors.initials: S
authors.lastname: Li
authors.forename: Lei
authors.initials: L
authors.lastname: Yang
authors.forename: Jin
authors.initials: J
authors.lastname: Huang
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D058851:GPI-Linked Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D009414:Nerve Growth Factors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects; D009479:Neuropeptides / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
chemical_list: D058851:GPI-Linked Proteins; C484539:NRN1 protein, human; D009414:Nerve Growth Factors; D009479:Neuropeptides; D011994:Recombinant Proteins