Dai Xiaoyong, Sun Zhongqing, Liang Rui, Li Yu, Luo Huanmin, Huang Yadong, Chen Meiwan, Su Zhijian, Xiao Fei
Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, 510632, People's Republic of China.
Appl Microbiol Biotechnol. 2015 Jul;99(14):5997-6007. doi: 10.1007/s00253-015-6477-5. Epub 2015 Mar 12.
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.
Nogo-66是Nogo-A C末端两个疏水结构域侧翼的一段由66个氨基酸组成的亲水环，它与Nogo-66受体（NgR）相互作用，在中枢神经系统（CNS）中发挥多种功能。Nogo-66在神经元发育的多个方面发挥重要作用，包括细胞迁移、轴突导向、成束以及树突分支，并且在CNS可塑性的多个方面也有作用，包括少突胶质细胞分化和髓鞘形成。在此，将小泛素相关修饰物（SUMO）与目标基因Nogo-66融合，并在大肠杆菌（E. coli）中表达该构建体。在最佳发酵条件下，融合蛋白的可溶性表达水平占总上清蛋白的33%。用SUMO蛋白酶切割融合蛋白并通过镍-氮三乙酸（Ni-NTA）亲和层析进行纯化后，10升规模发酵获得的重组Nogo-66的产量和纯度分别为23±1.5毫克/升和大于93%。通过电喷雾电离质谱分析确认了重组Nogo-66的真实性。功能分析表明，重组Nogo-66能够特异性结合NgR。免疫荧光结果显示，重组Nogo-66能够显著抑制神经生长因子刺激的大鼠嗜铬细胞瘤（PC12）细胞和小脑颗粒细胞（CGC）的神经突生长。此外，Nogo-66通过增加磷酸化的含 Rho相关卷曲螺旋结构域蛋白激酶2（ROCK2）、塌陷反应介导蛋白2（CRMP2）和肌球蛋白轻链（MLC）的水平来抑制神经突生长。本研究提供了一种可行且便捷的生产方法，可产生足够量的重组Nogo-66用于实验和临床应用。
