Recombinant Nogo-66 via soluble expression with SUMO fusion in Escherichia coli inhibits neurite outgrowth in vitro

Applied Microbiology and Biotechnology
Xiaoyong DaiFei Xiao

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 ...Continue Reading

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Citations

Dec 30, 2015·Molecular and Cellular Neurosciences·Zhongqing SunFei Xiao
Mar 28, 2019·Molecular Neurobiology·Aubin MoutalRajesh Khanna
Jan 27, 2020·Analytical Biochemistry·Trivikram R MoluguJoe G N Garcia

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