Overexpression and Purification of Arabidopsis thaliana HKT1;1 protein in yeast Pichia pastoris

Abstract

Salinity is one of the most important abiotic stresses on about 20% of the world's irrigated areas. It is a serious issue that compromises world food production. Salt tolerance in plants depends on the regulation of ion homeostasis by membrane transporters AtHKT1;1 of Arabidopsis thaliana is one such crucial transporter. It helps in the retrieval of Na⁺ from the xylem sap to safeguard photosynthetic tissues. AtHKT1;1 has been physiologically validated in several organisms, but there is no report of its functionality in Pichia pastoris, a scalable platform that is ideal for biochemical and structural studies. The purpose of this thesis was to heterologouslyexpress, purify and detect AtHKT1;1 in Pichia pastoris GS115. Transformed cells with the pPICZ-A‑athkt1;1construct was grown in BMGY medium, induced with methanol for 72 h and harvested. Two parallel membrane isolation protocols were tested: direct Sonication followed by solubilization and Zymolyase mediated spheroplast formation followedby Sonication and solubilization. His‑tagged protein was prepared using Ni‑NTA affinity column and eluted with 300 mM imidazole. The level of protein expression and purification was tracked by SDS-PAGE and Western blotting with anti‑His and anti‑eGFP antibodies. Elution fractions from both isolation techniques showed a band corresponding to the expected molecular weight on Coomassie-stained gels and Western blots, and the isolation and purification of AtHKT1;1 in Pichia pastoris were successful. The purified protein was kept at –80 °C for future studies. This work represents the first functional validation of AtHKT1;1 produced in P. pastoris and includes a reproducible pipeline for production of AtHKT1;1 that enables further analysis, including interactome mapping and structural characterization.