PROPERTIES AND PURIFICATION OF AN ACTIVE BIOTINYLATED LACTOSE PERMEASE FROM ESCHERICHIA-COLI

A simplified approach for purification of functional lactose permease from Escherichia coli is described that is based on the construction o f chimeras between the permease and a 100-amino acid residue polypepti de containing the biotin acceptor domain from the oxaloacetate decarbo xylase of Klebsiella pneumoniae Cronan, J. E., Jr. (1990) J. Biol. Ch em. 265, 10327-10333!. Chimeras were constructed with a factor Xa prot ease site and the biotin acceptor domain in the middle cytoplasmic loo p (loop 6) or at the C terminus of the permease. Each construct cataly zes active lactose transport in cells and right-side-out membrane vesi cles. Moreover, the constructs are biotinylated in vivo, and in both c himeras, the factor Xa protease site is accessible from the cytoplasmi c surface of the membrane. Both biotinylated permeases bind selectivel y to immobilized monomeric avidin and are eluted with free biotin in a high state of purity, and the loop 6 chimera catalyzes active transpo rt after reconstitution into proteotiposomes. The methodology describe d should be applicable to other membrane proteins.