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.
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Acknowledgments
This study was supported by the Natural Science Foundation of China (81202519 and 81403120) and Guangdong Province (S2011040002140), by the Macao Science and Technology Development Fund (102/2012/A3), Fundamental Research Funds for the Central Universities Project (21611378), supported by Guangdong Province Higher Vocational Colleges and Schools Pearl River Scholar Funded Scheme (2012) (21613408) and Ocean and Fisheries Foundation of Guangdong Province (A201301B09). The authors are grateful to Minjing Zhang, Xue Xiao, and Zhijian Wu for the technical assistance.
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Xiaoyong Dai and Zhongqing Sun contributed equally to this work.
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Dai, X., Sun, Z., Liang, R. et al. Recombinant Nogo-66 via soluble expression with SUMO fusion in Escherichia coli inhibits neurite outgrowth in vitro. Appl Microbiol Biotechnol 99, 5997–6007 (2015). https://doi.org/10.1007/s00253-015-6477-5
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DOI: https://doi.org/10.1007/s00253-015-6477-5