Screening And Expression For Voltage-Gated Sodium Channel Inhibitors

The voltage-gated sodium channels(VGSCs)are distributed in nervous system.A lot of neuronal pathologies,such as epilepsy and chronic pain are related to the dysfuction of voltage-gated sodium channels.Selecting proper inhibitors of VGSCs can alleviate the symptoms effectively.Most peptides,possessing the structure of inhibitor cystine Knot(ICK),are capable of resistance of extremes of pH,high temperature and proteases.Moreover,voltage-gated sodium channels have some relationships with the disorder of neuronal excitability.Which make them being the useful pharmacological tool.ICK toxins can modulate the activities of voltage-gated sodium channels,which has been linked to disorder of neuronal excitability.Isolating and purification of peptides from venom are costing and time-consuming.The spider-venom peptide Hainantoxin-Ⅳ(HNTX-Ⅳ)is a typical ICK molecule which possesses three disulfide bonds.In this study,a random DNA library was constructed,based on five critical residues of Hainantoxin-Ⅳ being randomized.The yeast two-hybrid strategy provides a high-throuhput method,which was often used in the study of protein-protein interactions.In this study,the extracellular segment DⅡ-S1-S2 of Nav1.3 was used as a bait protein to screen this random library.Two DNA sequences were identified and marked as R1 and R2 after excluding the false positive.It was demonstrated that R1 and R2 have no active effects.Further study based on molecular modeling demonstrated that the five critical residues of R1 and R2 were technically feasible in yeast two-hybrid.According to previous study,JZTX-Ⅲ has no detectable interaction effect on the extracellular segment DⅡ S1-S2 of Nav1.3 using by yeast two-hybrid assay.However,by transplanted the critical residues of R1 or R2 to JZTX-Ⅲ,interactions could be detected by yeast two-hybrid assay.Generally,peptides can be obtained by two methods,chemical synthesis or hetero-prokaryotic expression.The disadvantages of chemical synthesis are low yields,high costs,and even no natural bioactivity.For this reason,prokaryotic expression turned out to be the best choice during later study.The target gene was inserted into the expression vector pET43 and E.coli BL21(DE3)was chosen to be a host strain to obtain R1andR2 small peptides.In order to avoid the formation of insoluble inclusion bodies,GST fusion tag,which has solubilization effect was inserted into the N-terminal of target protein.After IPTG induction,the expression of the fusion protein was soluble and can be affiliated by GST resin.SUMO has became an effective fusion and purification tag for protein expression.When the target protein was fused to the C-terminal of SUMO,there was a cleavage site between target protein and resultant fusion protein.In subsequent experiments,the prepurified fusion protein was cleaved by SUMO protease,and the target protein was detected by SDS-PAGE.The results demonstrated that removing GST and SUMO into the N-terminal of target protein and building a cleavage site with the expression vector pET43 was a good prokaryotic expression method.