Expression And Purification Of ω-CTX M ⅦA

[Backgrounds]Cone snails (genus Conus) have more than 500 species. Each of them contains 50 to 200 different toxic peptides in the venom. Most Conus peptides rich in cysteine residues which can form multiple disulfide bonds have been referred to as conotoxins (CTXs). In terms of the pattern of disulfide linkages and signal sequences, conotoxins can be divided into four large groups: O-, A-, M- and T-superfamilies. Most of conotoxins are able to affect different ion channels and widely used as molecular probes in neuroscience research as well as diagnostic and therapeutic purposes.-Conotoxins which belong to O-superfamily share a conserved disulfide framework and target to voltage-sensitive calcium channels (VSCCs). -CTX M VIIA, first isolated from the venom of Conus magus, is a hydrophilic peptide containing 25 amino acids with a net charge of +6. Three rigid intramolecular disulfide bridges between the conserved cysteine residues (Cl and C16, C8 and C20, C15 and C25 ) allow the peptide to fold into a compact globular conformation containing four different loops. Several lines of evidence indicate that the backbone is crucial for its biological function. -CTX M VIIA is a potent and specific blocker of N-type calcium channels. Many studies have demonstrated that -CTX M VIIA has effects of antinociception and neuroprotection by intrathecal and spinal administration. The mechanism of analgesia is that the conotoxin blocks neurotransmitter release fromnociceptive afferents thereby inhibiting the pain signal propagation to the brain (by spinal injection) or directly targets nociceptive neurotransmission (by intrathecal injection). The neuroprotective function is related to its blockage of excitotoxic neurotransmitter release and arrestment of the pathologic calcium entry into neurons after ischemia and trauma to the brain. The conotoxin has demonstrated efficacy in animal models of global, focal cerebral ischemia and pain syndromes.Encouraged by the double effect of antinociception and neuroprotection , we were interested in producing -CTX M VDA by gene engineering procedure. In this paper, we constructed a recombinant prokaryotic expression plasmid with the fusion partner of Trx and obtained the fusion protein Trx-CTX with analgesic activity . [Methods]An artificial DNA sequence encoding co-CTX M VHA was designed by using E coli-like codons. The double-stranded DNA was synthesized by ligation of four fragments. Kpn I and enterokinase sites were introduced at the 5' terminus whereas a stop codon and EcoR. I site were added at 3' terminus. The synthetic DNA fragments were phosphorylated by T4 PNK. Before ligation, the single stranded fragments were annealed to form the paired double strands. The equal amount of fragments CTX1 and CTX2 were mixed and incubated at 95 . The mixture was cooled down to 45 gradually at room temperature. Another paired fragments CTX3 and CTX4 were treated in same procedures. The two annealed DNA strands were ligated by T4 DNA ligase. Finally, the full gene fragment was cloned into pET-32a(+) cut with Kpn I/ EcoR I. The recombinant plasmid pET-32a(+)-CTX M VIIA was checked by restriction enzyme digestion and was then subjected to DNA sequencing to confirm the desired gene sequence.E coli BL21(DE3) transformed with the expression vector was grown at 37掳C until the optical density reached 0.8. Expression was induced by addition of IPTG to a final concentration of 1 mM and incubation for 4 h. The bacteria were then collected by centrifugation.The His-tag in the fusion protein Trx-CTX allowed it to be purified by Metal Chelated Affinity Chromatography (MCAC) on Akta Purifier (Amersham). After resuspended in buffer, the collected bacteria were sonicated twice in the present of 0.05% Triton X-100. Then the bacterial lysate was centrifuged and the supernatant containing the soluble fusion protein was applied to a Cu-IDA Chelated Fast Flow column. The expected fusion protein was eluted with series of buffers in order. Subsequently gel filtration on a Coarse Fast Flow G-25 colum...