High-yield expression and functional analysis of Escherichia coli glycerol-3-phosphate transporter.
The glycerol-3-phosphate (G3P) transporter, GlpT, from Escherichia coli mediates G3P and inorganic phosphate exchange across the bacterial inner membrane. It possesses 12 transmembrane alpha-helices and is a member of the Major Facilitator Superfamily. Here we report overexpression, purification, and characterization of GlpT. Extensive optimization applied to the DNA construct and cell culture has led to a protocol yielding approximately 1.8 mg of the transporter protein per liter of E. coli culture. After purification, this protein binds substrates in detergent solution, as measured by tryptophan fluorescence quenching, and its dissociation constants for G3P, glycerol-2-phosphate, and inorganic phosphate at neutral pH are 3.64, 0.34, and 9.18 microM, respectively. It also shows transport activity upon reconstitution into proteoliposomes. The phosphate efflux rate of the transporter in the presence of G3P is measured to be 29 micromol min(-1) mg(-1) at pH 7.0 and 37 degrees C, corresponding to 24 mol of phosphate s(-1) (mol of protein)(-1). In addition, the glycerol-3-phosphate transporter is monomeric and stable over a wide pH range and in the presence of a variety of detergents. This preparation of GlpT provides ideal material for biochemical, biophysical, and structural studies of the glycerol-3-phosphate transporter.
Auer
M
M
Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, New York 10016, USA.
Kim
M J
MJ
Lemieux
M J
MJ
Villa
A
A
Song
J
J
Li
X D
XD
Wang
D N
DN
D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003902:Detergents; D004355:Drug Stability; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005822:Genetic Vectors / Q000138:chemical synthesis; D005960:Glucosides; D005994:Glycerophosphates / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014364:Tryptophan / Q000378:metabolism
D002352:Carrier Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; C488962:GlpT protein, E coli; D005960:Glucosides; D005994:Glycerophosphates; D026901:Membrane Transport Proteins; D011510:Proteolipids; D011994:Recombinant Proteins; C021688:UgpB protein, E coli; C020485:proteoliposomes; C040358:dodecyl maltoside; D014364:Tryptophan; C029620:alpha-glycerophosphoric acid