The Expression,Purification And Activity Research Of N-type Calcium Ion Channel Inhibitor ?-conotoxin

?-cone snail toxin(Conopeptide, Conotoxin, CTX) is the first kind of natural toxin, which is identified to specifically block voltage-gated calcium channels(VGCC). M?A as a kind of ?-conotoxin, it can interact N-type voltage-gated calcium ion channels(NGCCs) specifically. NGCCs play an important role in the transmission of pain in the spinal cord, and MVIIA is able to block N-type voltage-gated calcium ion channels(Cav2.2) to achieve the analgesic effect, which has important clinical significance. But now M?A has severe side effects, including dizziness, nystagmus, somnolence, abnormal gait, and ataxia, which influences its wide application seriously. Recent researches have found that M?A mutant ?-2 has similar analgesia effects to M?A but with little side effects, so the synthesis of a large number of ?-2 toxin protein can greatly promote the ?-2 application to more research and medicine field. Up to now, the vast majority of polypeptide toxins include ?-2 toxin, which is mainly obtained by chemical methods such as solid-phase peptide synthesis, but the process is complex, time consuming and expensive. Therefore, in order to get a large number of ?-2 toxin efficiently and economically, this paper intends to develop a new method of expressing ?-2 toxin protein in E.coli expression system, and by this method we could obtain ?-2 toxin protein with high purity.?-2 toxin protein contains only 25 amino acid residues with abundant disulfide bond, and molecular weight is so small. All of them make it difficult to express and purify in E.coli cytoplasm which is a relatively partially reductive environment [1]. To acquire fusion protein of ?-2, we established the OrigamiB(DE3) cytoplasm expressed system which MBP-?-2 is co-expressed with Dsbc. In this system, the DsbC promotes the folding of ?-2 disulfide bond in right place. Through this system, we gain soluble and spatial structure folded correctly and highly purified ?-2 successfully. Finally, through the electrophysiological experiment, we detect that synthesized ?-2 is able to block Cav2.2 properly, so as to determine its physiological role.To sum up, I develop a novel method that could obtain ?-conotoxin M?A mutant ?-2 toxin protein in E.coli expression system successfully during the period of master, and this method provides a guarantee for the economical and efficient synthesis of a large number of ?-2, with certain economic and clinical application. At the same time, this work provides a reference for the batch synthesis of other similar peptide toxins, which can promote the research of biological polypeptide toxin in the field of drug application.