Purification and reconstitution of the glutamate carrier GltT of the thermophilic bacterium Bacillus stearothermophilus.
An affinity tag consisting of six adjacent histidine residues followed by an enterokinase cleavage site was genetically engineered at the N-terminus of the glutamate transport protein GltT of the thermophilic bacterium Bacillus stearothermophilus. The fusion protein was expressed in Escherichia coli and shown to transport glutamate. The highest levels of expression were observed in E. coli strain DH5 alpha grown on rich medium. The protein could be purified in a single step by Ni2+-NTA affinity chromatography after solubilization of the cytoplasmic membranes with the detergent Triton X100. Purified GltT was reconstituted in an active state in liposomes prepared from E. coli phospholipids. The protein was reconstituted in detergent-treated preformed liposomes, followed by removal of the detergent with polystyrene beads. Active reconstitution was realized with a wide range of Triton X100 concentrations. Neither the presence of glycerol, phospholipids, nor substrates of the transporter was necessary during the purification and reconstitution procedure to keep the enzyme in an active state. In B. stearothermophilus, GltT translocates glutamate in symport with protons or sodium ions. In membrane vesicles derived from E. coli cells expressing GltT, the Na+ ion dependency seems to be lost [Tolner, B., Ubbink-Kok, T., Poolman, B., & Konings, W. N. (1995) Mol. Microbiol. 18, 123-133], suggesting a role for the lipid environment in the cation specificity. In agreement with the last observation, glutamate transport catalyzed by purified GltT reconstituted in E. coli phospholipid is driven by an electrochemical gradient of H+ but not of Na+.
Gaillard
I
I
Department of Microbiology, Groningen Biotechnology and Biomolecular Sciences Institute, University of Groningen, The Netherlands.
Slotboom
D J
DJ
Knol
J
J
Lolkema
J S
JS
Konings
W N
WN
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D027322:Amino Acid Transport System X-AG; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011510:Proteolipids / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D011510:Proteolipids; C020485:proteoliposomes