In vitro biotinylation provides quantitative recovery of highly purified active lactose permease in a single step.
Consler et al. [Consler, T. G., Persson, B. L., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6934-6938] described a one-step purification of lactose permease, a hydrophobic membrane transport protein, from Escherichia coli. Permease constructs containing a biotin acceptor domain are biotinylated in vivo, followed by solubilization and avidin affinity purification. Although a high degree of purity is obtained, only about 15-20% of the permease is recovered due to incomplete biotinylation. In this communication, a simple modification is described that allows quantitative recovery of highly purified permease. Membranes containing permease with the biotin acceptor domain from the Klebsiella pneumoniae oxaloacetate decarboxylase are extracted with 5 M urea or treated with dicyclohexylcarbodiimide to inactivate F1/Fo ATPase and biotinylated in vitro with biotin ligase, ATP and d-biotin. Subsequently, the membranes are harvested, washed to remove free biotin and solubilized with 2% n-dodecyl-beta-D-maltopyranoside. Biotinylated permease is then purified in one step by affinity chromatography on monomeric avidin-Sepharose. The purified material is homogeneous and exhibits full activity with respect to ligand binding and counterflow.
Pouny
Y
Y
Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California, Los Angeles 90095-1662, USA.
Weitzman
C
C
Kaback
H R
HR
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport / Q000235:genetics; D019921:Biotinylation; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D029968:Escherichia coli Proteins; D008024:Ligands; D008081:Liposomes / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D013050:Spectrometry, Fluorescence; D027981:Symporters
D000817:Anilino Naphthalenesulfonates; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D008081:Liposomes; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; C021499:lactose permease