Heterologous over-“expression” of membrane proteins (Trks) in Pichia pastoris
TRK proteins are cation translocating proteins that allows S. cerevisiae (S. c.) and other yeast to survive and grow under different environment from a few µM to hundreds of mM [K+] while maintaining internal K+ concentration relatively constant. In S. c. there are two specific K+ translocation systems: Trk1 and Trk2. Trk1 is 1235 amino acids long and Trk2 is 889 amino acid long. A unique feature of fungal Trks is that they contain a “Long Hydrophilic Loop” (LHL) which is not homologous between these proteins and differs largely in length i.e. 648 aa in Trk1 and 327 aa in Trk2. The function of LHL is unknown. The two-main yeast expression systems which are widely used for membrane proteins are S. cerevisiae and Pichia Pastoris. In this project we plan to construct different plasmids from pPINK-HC (a high copy number plasmid) carrying full length TRK1, TRK2, TRK1ΔLHL and TRK2ΔLHL with 6xHis tag and fused with GFP (fusing with GFP will enable us to visualize protein microscopically and to quantify the amount of protein produced using spectrophotometer). Three protease knockout strains along with the “protease wild-type” PichiaPink strain will be transformed with each of the above-mentioned plasmids. Transformants will be analysed by PCR to check the presence of the TRK-fusion genes. Heterologous overexpression in PichiaPink strains is driven by (the alcohol-oxidase) AOX1 promoter hence methanol induction studies will be performed. Trk protein expression will be analysed by Coomassie-stained SDS-PAGE and western blot using anti-GFP antibodies.