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 of chimeras between the permease and a 100-amino acid residue polypeptide containing the biotin acceptor domain from the oxaloacetate decarboxylase of Klebsiella pneumoniae [Cronan, J. E., Jr. (1990) J. Biol. Chem. 265, 10327-10333]. Chimeras were constructed with a factor Xa protease site and the biotin acceptor domain in the middle cytoplasmic loop (loop 6) or at the C terminus of the permease. Each construct catalyzes active lactose transport in cells and right-side-out membrane vesicles. Moreover, the constructs are biotinylated in vivo, and in both chimeras, the factor Xa protease site is accessible from the cytoplasmic surface of the membrane. Both biotinylated permeases bind selectively to immobilized monomeric avidin and are eluted with free biotin in a high state of purity, and the loop 6 chimera catalyzes active transport after reconstitution into proteoliposomes. The methodology described should be applicable to other membrane proteins.
Consler
T G
TG
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1662.
Persson
B L
BL
Jung
H
H
Zen
K H
KH
Jung
K
K
Privé
G G
GG
Verner
G E
GE
Kaback
H R
HR
D001426:Bacterial Proteins / Q000302:isolation & purification; D001692:Biological Transport; D001710:Biotin; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D007785:Lactose / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification; D026901:Membrane Transport Proteins / Q000302:isolation & purification*; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D027981:Symporters
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D027981:Symporters; D001710:Biotin; C021499:lactose permease; D007785:Lactose