| The genus Conus are marine meat-eating Mollusks.They belong to Gastropod and Conidae, there are nearly 500 all the world. Conopeptides (conotoxins. CTXs) obtained from Conus are the important kind of bioactive neurotoxin peptides. There are about 100～200 different kinds of conotoxins in each Conus, which means that, there are more than 50000 kinds of conotoxins in the world. Usually the conotoxins are consisted of no more than 40 amino acid residues with disulfide-rich, novel chemical structure, strong biological activity and high selectivity on ion channels or other receptors. So far, they have been categorized into several superfamilies named O, A, I, M, P, S, and T etc., which are characterized by the number of their cysteine residues. Conopeptides have been unprecedented attention in the field of neuroscience research and drug development aspects, so it is significant to research conotoxins.The protein disulfide isomerase (PDI) is an important foldase in folding of the nascent polypeptide chain lay in the endoplasmic reticulum (ER). The venom of tropic officinal marine cone snails is abundant with PDI which is important in assisting the folding and maturation of secretory conopeptides in vivo. The results were shown as follows:(1).Pure natural PDI was obtained from the venom of Conus betulinus native to Hainan mainly by Gel-filtration chromatography and Rotofor preparative liquid-phase isoeletric focusing electrophoresis, which was confirmed by SDS-PAGE and MALDI-TOF Mass Spectrometry. An efficient protocol for natural Conus PDI purification was set up. The activity of PDI was tested by using linear conopeptide K412 as substrate, showing that the PDI could catalyze oxidative folding of K412.(2).We cloned three new PDI gene respectively from Conus textile, C. betulinus and C. lividus natived to Hainan by PCR method. Then the PDI genes were subcloned for relevant restriction Enzyme cutting site, so they can connect with pET expression vectors to be new prokaryotic expression vector. We successfully constructed 4 expression vectors about PDI gene:pET22b(+)-PDI42, pET22b(+)-PDI49, pET28b(+)-PDI12 and pET28b(+)-PDI19.(3).Mature peptide genes of the O-superfamily conotoxins Ma6A and Ma6B was synthesized chemically. Each conotoxin has two types of mature peptide genes:with termination codon or not, means the corresponding peptides without His-Tag or with His-Tag.4 expression vectors containing conotoxin Ma6A/B gene were constructed successfully:PA2,PB2,PA3 and PB3.(4).4 kind of expression vectors pET22b(+)-PDI42, PA2, PA3 and PB3 expressed successfully in E.coli BLR21(DE3).Oxidative folding of conopeptides is very complex. Only conopeptides with correct disulfide bond connections are active in various pharmacology. The result of this work would be the important basis for further research on mechanism and application of PDI in the oxidative folding of a great variety conopeptides. |
