Recombinant Expression In Pichia pastoris System Of The Potassium Channel Inhibitor Peptide, Ts6.

Panda, Lipsa Rani
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The therapeutic potential of many potent venomous peptides is known to block ion channels, yet diseases that have a scope of being treated have not been discussed. In this study, Ts6 which has proven blocking activity in Kv1.3 has been analyzed recombinantly for Kv1.5 and Kv1.3. This toxin (Ts6) purified from Tityus serrulatus venom (a Brazilian scorpion) is composed of 40 amino acid residues, four disulfide bonds, and a molecular weight of 4.5 kDa. In the scope of this research, we used X-33 Pichia pastoris yeast which produced Ts6 extracellularly in substantial amount. The plasmid vector pPICZαA-Ts6 was constructed, where the N-terminal was his-tagged and an enterokinase cleavage site was inserted. The X-33 Pichia clones were cultured in increasing concentration of ZeocinTM. After methanol induction, protein expression yielded around 4.7 mg/l Ts6. The purification of the his-tag Ts6 was done in two steps that included the Ni2+-NTA affinity chromatography then followed by reversed-phase-HPLC. Protein was quantified using bicinchoninic acid (BCA) assay to know the protein yield concentration. Proteolytic cleavage of enterokinase site was done and untagged Ts6 was reached. Electrophysiological recordings were done on Kv1.3 and Kv1.5. Henceforth, present study reports the successful production of recombinant Ts6 protein in Pichia pastoris expression system that showed significant effect in inhibiting potassium channels.
Toxins, Affinity chromatography, Ts6, Kv 1.5, RP-HPLC, Electrophysiology, Protein production & purification, his tag removal, PCR, SDS PAGE