Biological Synthesis Of Two Conotoxins Ma6A And MaI126

The genus Conus belong to Gastropod Mollusks, and there are nearly 500 in the world. Conotoxins (conopeptides, CTXs) are one kind of the bioactive peptide toxins which are obtained from the genus. Recent researches suggest that, there may be 1000-1900 kinds of conotoxins in each Conus, namely, there are more than 500000 kinds of conotoxins all of the world, which substantially exceeds preciously known about five million. Usually the conotoxins are consisted of 8-40 amino acid residue with disulfide-rich, novel chemical structure, strong biological activity and high selectivity on ion channels or receptors. So far they have been categorized into several superfamilies named A, I, M, O, P, S, and T etc., which are characterized by the number of their cysteine residues. They have been unprecedented attention in the field of neuroscience research and drug development aspects, so it is significant to research conotoxins. Research and development of contoxins depends on chemical synthesis mainly due to rare natural conotoxins. However there are some disadvantages of peptide chemical synthesis, such as. It’s more difficult to compose the mixture for its high toxicity,low production rate,low purity and high cost, especially for the conopeptides with larger molecular weight. Synthesized linear conopeptides needs to be folded cumbersomely for getting bioactive peptides with correct disulfide bonds. Therefore, development of a large number of conotoxins can not depend on the chemical synthesis alone, which can not meet requirements as a drug research and commercial production. It is known that one conopeptide in the Conus cells are product of its precursor gene expression and post-translational modification, so large amount of one conopeptide could be obtained by new valid biosynthesis method of the recombinant conopeptide gene expression.This research explores biosynthesis method of two conopeptides Ma6Aand MaI126 with low cost. Both genes of Ma6A and MaI126 are from Conus marmoreus which belong to O-and T-superfamily respectively. The Ma6A mature peptide is composed of 31 amino acids with three disulphide bonds which is an antagonist of mammal sodium channels; while the MaI126 is composed of 13 amino acids with two disulphide bonds. The eukaryote expression system of Pichia pastoris was used to produce recombinant conotoxins Ma6A and MaI126. We hope to get the recombinant peptides with natural configuration through Pichia pastoris intracellular posttranslational modifications system, then the recombinant conotoxins finished the oxidation folding and secreting into medium during this procession, which could be purified later.Two yeast expression vectors pPICZaA-Ma6Af and pPICZaA-Ma6 were constructed with Ma6A mature peptide gene and yeast signal peptide DNA, which contained or did not contain protein purification label. Another yeast expression vector pPICZA-MaI126 with Ma6A mature peptide gene and its own signal peptide DNA was constructed in our lab before. All the three recombinant plasmids were linearized, and integrated into Pichia pastoris chromsome by electroporation. The yeast recombinants were screened by Zeocin antibiotic resistance, which were identified by target genes’PCR amplification and sequencing the PCR products cloned in T-vector. The yeast recombinants with full-length target gene and high copies were selected for expression which was induced by methanol.We explored and optimized the inducing conditions and matches between carriers and the host bacteria. The results showed that the expression of Ma6A was induced with 0.5% methanol as final concentration every 72h, the MM medium is the best one, the hostⅩ-33 was better than the other two hosts GS115, KM71. The supernatant total protein was analized by Tricine-SDS-PAGE, in which there are two target proteins appeared with 6kD for fusion Ma6A and 3.5kD for natural peptide Ma6A. The recombinant conotoxin MaI126 with molecular weight 1.4 kD were identified by RP-HPLC, which need to be purified and bioassayed later. Therefore, we have developed an efficient and functional expression system for Ma6A and MaI126 with a new valid biosynthesis method, which would not only facilitate further studies of conotoxin genes’function, but also allow possible large-scale production of conotoxin clinic drugs in the future.