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

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.

A novel Nogo-66 receptor antagonist peptide promotes neurite regeneration in vitro

“…To investigate the neurite promoting activity of NAP2, we utilized two neuronal cell types, PC12 cells and CGCs in vitro, employing Nogo-66 as a neurite outgrowth inhibitory protein (Dai et al, 2015). PC12 cells and CGCs have been widely used to study neurite growth inhibition and promotion by various ligands, including Nogo-66, OMgp (oligodendrocyte-myelin glycoprotein) and MAG (myelin-associated glycoprotein).…”

“…NgR1 was obtained from R&D Systems (McKinley, Minneapolis, USA). Nogo-66 was obtained via soluble expression with SUMO fusion in E. coli (Dai et al, 2015) from our own lab. The horseradish peroxidase (HRP)-anti-M13 mAb was from Amersham Pharmacia Biotech (Uppsala, Sweden) and the horseradish peroxidase (HRP)-streptavidin was from Invitrogen corporation (California, USA).…”

Nogo‑66 promotes β‑amyloid protein secretion via NgR/ROCK‑dependent BACE1 activation

“…Reagents. Soluble Nogo-66 protein was obtained via SUMO fusion in Escherichia coli from our laboratory (16). Y-27632 and the NEP1-40 were obtained from Merck KGaA and Tocris Bioscience, respectively.…”

Expression and purification of antimicrobial peptide AP2 using SUMO fusion partner technology inEscherichia coli

“…; Dai et al . ). The important advantage of the SUMO fusion technology is that it generates peptides with a native N‐terminus without residual amino acids after cleavage with SUMO protease (Malakhov et al .…”