title: Recombinant Nogo-66 via soluble expression with SUMO fusion in Escherichia coli inhibits neurite outgrowth in vitro.
abstract: Nogo-66, a hydrophilic loop of 66 amino acids flank two hydrophobic domains of the Nogo-A C terminus, interacts with the Nogo-66 receptor (NgR) to exert numerous functions in the central nervous system (CNS). Nogo-66 has important roles in aspects of neuronal development, including cell migration, axon guidance, fasciculation, and dendritic branching, and in aspects of CNS plasticity, including oligodendrocyte differentiation and myelination. Here, the small ubiquitin-related modifier (SUMO) was fused to the target gene, Nogo-66, and the construct was expressed in Escherichia coli (E. coli). Under the optimal fermentation conditions, the soluble expression level of the fusion protein was 33 % of the total supernatant protein. After cleaving the fusion proteins with SUMO protease and purifying them by Ni-NTA affinity chromatography, the yield and purity of recombinant Nogo-66 obtained by 10-L scale fermentation were 23 ± 1.5 mg/L and greater than 93 %, respectively. The authenticity of the recombinant Nogo-66 was confirmed by an electrospray ionization-mass spectrometry analysis. The functional analyses indicated that the recombinant Nogo-66 was capable of binding the NgR specifically. The immunofluorescence results showed that the recombinant Nogo-66 could significantly inhibit neurite outgrowth of rat pheochromocytoma (PC12) cells stimulated by nerve growth factor and cerebellar granule cells (CGCs). Furthermore, Nogo-66 inhibited neurite outgrowth by increasing the level of phosphorylated Rho-associated coiled-coil-containing protein kinase 2 (ROCK2), collapsin response mediator protein 2 (CRMP2), and myosin light chain (MLC). This study provided a feasible and convenient production method for generating sufficient recombinant Nogo-66 for experimental and clinical applications.
authors.affiliation: Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, 510632, People's Republic of China.
authors.forename: Xiaoyong
authors.initials: X
authors.lastname: Dai
authors.forename: Zhongqing
authors.initials: Z
authors.lastname: Sun
authors.forename: Rui
authors.initials: R
authors.lastname: Liang
authors.forename: Yu
authors.initials: Y
authors.lastname: Li
authors.forename: Huanmin
authors.initials: H
authors.lastname: Luo
authors.forename: Yadong
authors.initials: Y
authors.lastname: Huang
authors.forename: Meiwan
authors.initials: M
authors.lastname: Chen
authors.forename: Zhijian
authors.initials: Z
authors.lastname: Su
authors.forename: Fei
authors.initials: F
authors.lastname: Xiao
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D036341:Intercellular Signaling Peptides and Proteins; D009185:Myelin Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016501:Neurites / Q000187:drug effects*; D000070798:Nogo Proteins; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D009185:Myelin Proteins; D018994:Myosin Light Chains; D009419:Nerve Tissue Proteins; D000070798:Nogo Proteins; D011993:Recombinant Fusion Proteins; C000603197:Rtn4 protein, rat; D025842:SUMO-1 Protein; C095114:collapsin response mediator protein-2; C580818:ROCK2 protein, rat; D054460:rho-Associated Kinases