RAPID PURIFICATION OF A FUNCTIONALLY ACTIVE-PLANT SUCROSE CARRIER FROM TRANSGENIC YEAST USING A BACTERIAL BIOTIN ACCEPTOR DOMAIN

A rapid and efficient method has been used for the purification of a P lantago major sucrose carrier from Saccharomyces cerevisiae. The C-ter minal fusion of a bacterial biotin acceptor domain to the carrier prot ein did not interfere with the targeting to the yeast plasma membrane nor with the catalytic activity of the sucrose carrier. The chimeric c onstruct is biotinylated by yeast cells in vivo and represents the onl y biotinylated protein in yeast membranes. Solubilized biotinylated ca rrier protein binds selectively to immobilized monomeric avidin and ca n be eluted as pure protein with free biotin. The purified protein is functionally active and catalyzes the energy-dependent transport of su crose into proteoliposomes.