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Recombinant Nogo-66 via soluble expression with SUMO fusion in Escherichia coli inhibits neurite outgrowth in vitro
Dai X.1; Sun Z.1; Liang R.1; Li Y.1; Luo H.1; Huang Y.1; Chen M.2; Su Z.1; Xiao F.1
2015-07-26
Source PublicationApplied Microbiology and Biotechnology
ISSN14320614 01757598
Volume99Issue:14Pages:5997-6007
AbstractNogo-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.
KeywordBioassay Expression Nogo-66 Purification Small ubiquitin-related modifier
DOI10.1007/s00253-015-6477-5
URLView the original
Language英語
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Cited Times [WOS]:3   [WOS Record]     [Related Records in WOS]
Document TypeJournal article
CollectionInstitute of Chinese Medical Sciences
Affiliation1.Jinan University
2.Universidade de Macau
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GB/T 7714
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[J]. Applied Microbiology and Biotechnology,2015,99(14):5997-6007.
APA Dai X..,Sun Z..,Liang R..,Li Y..,Luo H..,...&Xiao F..(2015).Recombinant Nogo-66 via soluble expression with SUMO fusion in Escherichia coli inhibits neurite outgrowth in vitro.Applied Microbiology and Biotechnology,99(14),5997-6007.
MLA Dai X.,et al."Recombinant Nogo-66 via soluble expression with SUMO fusion in Escherichia coli inhibits neurite outgrowth in vitro".Applied Microbiology and Biotechnology 99.14(2015):5997-6007.
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