RESUMO
Recombinant expression systems for mammalian membrane transport proteins are often limited by insufficient yields to support structural studies, inadequate post-translational processing and problems related with improper membrane targeting or cytotoxicity. Use of alternative expression systems and optimization of expression/purification protocols are constantly needed. In this work, we explore the applicability of the laboratory strain LEXSY of the ancient eukaryotic microorganism Leishmania tarentolae as a new expression system for mammalian nucleobase permeases of the NAT/NCS2 (Nucleobase-Ascorbate Transporter/Nucleobase-Cation Symporter-2) family. We achieved the heterologous expression of the purine-pyrimidine permease rSNBT1 from Rattus norvegicus (tagged at C-terminus with a red fluorescent protein), as confirmed by confocal microscopy and biochemical analysis of the subcellular fractions enriched in membrane proteins. The cDNA of rSNBT1 has been subcloned in a pLEXSY-sat-mrfp1vector and used to generate transgenic L. tarentolae-rsnbt1-mrfp1 strains carrying the pLEXSY-sat-rsnbt1-mrfp1 plasmid either episomally or integrated in the chromosomal DNA. The chimeric transporter rSNBT1-mRFP1 is targeted to the ER and the plasma membrane of the L. tarentolae promastigotes. The transgenic strains are capable of transporting nucleobases that are substrates of rSNBT1 but also of the endogenous L. tarentolae nucleoside/nucleobase transporters. A dipyridamole-resistant Na+-dependent fraction of uptake is attributed to the exogenously expressed rSNBT1.
