Szerző szerinti böngészés "Pham Tran Nguyet, Ha"

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    Recombinant expression of the promiscuous ion channel inhibitor peptide, Hcr 1b-2, using Pichia pastoris expression system
    Pham Tran Nguyet, Ha; Papp, Ferenc; Borrego Terrazas, Jesús Angel; DE--Természettudományi és Technológiai Kar--Biotechnológiai Intézet
    Animal venom is a veritable treasure mine of active peptides, proteins, and neurotransmitters, among other components. Many potent peptides have been shown to have therapeutic potential blocking ion channels, however, diseases that can be treated have not been discussed. The characteristics of the recombinant Hcr 1b-2, which has demonstrated blocking activity on several ion channels, were analyzed in this study. This native toxin (Hcr 1b-2) was isolated from the sea anemone Heteractis crispa. It is made up of 41 amino acid residues, and has a molecular mass of 5238 Da. In the scope of this research, we used Pichia pastoris X-33 yeast to produce Hcr 1b-2 peptide in recombinant form. Several techniques were used to achieve this aim: the construction of expression cassette pPICZαA Hcr 1b-2 was done using enzymatic restriction digestion and ligation; transformation and clones’ selection processes where the Pichia X-33 clones were cultured in increasing concentrations of Zeocin™; expression screening and protein production using shake-flask; the purification of the peptide was done in two phases: affinity chromatography and reverse phase - high-performance liquid chromatography; lastly, the effect on the KV1.3 channel was tested through electrophysiological assays. As a result, the current work describes the effective production of the recombinant protein Hcr 1b-2 in Pichia pastoris expression system, which demonstrated a suppressing potassium ion channel activity.