Cloning,prokaryotic Expression And Purification Of Toxins From Centipede Scolopendra Subspinipes Mutilans

Scolopendra subspinipes mutilans is widely distributed in China,and there are some studies about its venoms.Currently,many peptide neurotoxins are isolating and identificating from the venoms to be found acting on voltage-gated ion channels.?-SLPTX-Ssm2 a,a kind of peptide toxin of Scolopendra,can depress the K+ current amplitude by 45% as the concentration is 200 nM.By searching NCBI database,series of toxins,?-SLPTX-Ssm2 b,?-SLPTX-Ssm2 c,?-SLPTX-Ssm2 d and ?-SLPTX-Ssm2 e,are found to have a very similar structure of ?-SLPTX-Ssm2 a,four 31-residus peptides with six Cys residues forming three intramolecular disulfide bridges.The coding squences are obtained by gene synthesis after codon optimization.The ?-SLPTX-Ssm2 b and ?-SLPTX-Ssm2 d are inserted to the expression vector pET-His-SUMO by RF-cloning and the ?-SLPTX-Ssm2 c and ?-SLPTX-Ssm2 e to the expression vector pET-GST-SUMO.Expressed with auto-induction in the E.coli strain BL21(DE3),the fusion proteins are firstly purified by affinity chromatography,Ni-NTA resin column is used to purified the fusion proteins contain 6?His tag,and GST resin column to GST tag.After desalted and concentrated by ultrafiltration,the toxins are released by digested through Ulp1 protease to cut off the SUMO tag.The released fragments are futher purified by RP-HPLC,and analyzed by MALDI-TOF mass spectrum assay.It was idetified that the moleculer weight of purified ?-SLPTX-Ssm2 b is 3390.4685 Da,identical to the theoretical one 3391.04 Da;and the moleculer weight of purified ?-SLPTX-Ssm2c(3451.773 Da)is identical to the theoretical one 3351.13 Da;the weight of ?-SLPTX-Ssm2 d,3390.8113 Da,is consistent to the theory,3391.89 Da;and the theorical molecular weight of ?-SLPTX-Ssm2 e is 3475.07 Da compared to the result 3475.732 Da by mass spectrum assay.It is demonstrated that the toxins are successfully expressied and purified to obtain three pairs of disulfide bridges from the experiment,and the method can be widely used to other peptide toxins contains many disulfide bridges.Next,the series of toxins we expressed will be detected to find their physiological activities,which lays a foundation to further screening of new insecticial peptide and developing new kind of drug.