Recombinant Nogo-66 via soluble expression with SUMO fusion in Escherichia coli inhibits neurite outgrowth in vitro
Recombinant Nogo-66 via soluble expression with SUMO fusion in Escherichia coli inhibits neurite outgrowth in vitro
复制标题
重组 Nogo-66 在大肠杆菌中通过可溶性表达与 SUMO 融合抑制体外神经突生长
DOI：
10.1007/s00253-015-6477-5
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发表时间：
2015-07-01
影响因子：
5
通讯作者：
Xiao, Fei
中科院分区：
文献类型：
--
作者：
Dai, Xiaoyong;Sun, Zhongqing;Xiao, Fei
Nogo-66是Nogo-A C末端两个疏水结构域侧翼的66个氨基酸的亲水环，与Nogo-66受体（NgR）相互作用以在中枢神经系统（CNS）中发挥多种功能。Nogo-66在神经元发育方面具有重要作用，包括细胞迁移、轴突导向、成束和树突分支，并且在CNS可塑性方面具有重要作用，包括少突胶质细胞分化和髓鞘形成。在这里，小泛素相关修饰物（SUMO）融合到靶基因Nogo-66，并在大肠杆菌（E. coli）中表达。大肠杆菌）。在优化的发酵条件下，融合蛋白的可溶性表达量占上清总蛋白的33%。经SUMO蛋白酶酶切和Ni-NTA亲和层析纯化后，10 L发酵获得的重组Nogo-66产量为23 ± 1.5 mg/L，纯度大于93%。重组Nogo-66的真实性通过电喷雾电离质谱分析确认。功能分析表明，重组Nogo-66能够与NgR特异性结合。 此外，Nogo-66通过增加磷酸化的Rho相关卷曲螺旋蛋白激酶2（ROCK 2）、肌球蛋白反应介导蛋白2（CRMP 2）和肌球蛋白轻链（MLC）的水平来抑制神经突生长。本研究为生产足够数量的重组Nogo-66提供了一种简便可行的生产方法。
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. 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.