Study On Gene Cloning, Expression And Purification Of Conotoxins

Conotoxins are biologically active peptides separated from venomous marine cone snails. They are typically small disulfide-rich peptides that potently and specifically interfere with neurotransmission by targeting a variety of proteins expressed on the cell surface,such as the ion channels of Ca²⁺, Na⁺ and K⁺, and the receptors of acetylcholine,NMDA,and neurotensin. So far they have been categorized into seven superfamilies named A, I, M, O, P, S, and T, which are characterized by the number of their cysteine residues, the arrangement of the disulfide bonds and the distinctive and conserved signal region of the toxin precursor. Conotoxins are utilized as reasearch tools in many fields of neurobiology with high-affinity antagonists for receptor and ion channel subtypes, and being small peptides, they are synthesizable and modifiable, thus increasing their availability and potential utility. As mentioned above they can be used both as pharmacological tools and as templates for drug design.In this study, four novel conotoxins κA-Ac4.1a/b, Mr6.1 and Mr5.2, belonging to A-, O-, T-superfamily respectively were cloned from C.marmoreus and C.achatinus with RACE. In addition, three novel conotoxins mr5.2, mr3.5 and mr3.6 were separated and purified from the venom of the mollusc-hunting cone snail C.marmoreus, and their primary structures were identified with mass-spectrometric and protein sequencing technologies. The mr5.2, which is belong to T-superfamily, exists with high degree of posttranslational modifications, contains two γ-carboxylation of glutamyl residuses. It is presumed that the Gla in conotoxins from invertebrate cone snail plays an important role for conotoxins folding. The other two, mr3.5 and mr3.6 have cysteine residue framework of CC-C-C-CC which is belong to O-spuperfamily, however, they were obviously different from the discoveried M-superfamily, the length of molecule is shorter, and there is only one amino acid in the third loop. They don't have GCxPC motif structure which exist in mr3a. Thus, the mr3.5 and mr3.6 were presumed to be a new family of M-superfamily.The fusion protein of pET-24(+)-PDI-Mr6.1 in E.coli BL21 was expressed with...