High-yield expression and functional analysis of Escherichia coli glycerol-3-phosphate transporter

被引:73
作者
Auer, M
Kim, MJ
Lemieux, MJ
Villa, A
Song, JM
Li, XD
Wang, DN
机构
[1] NYU, Sch Med, Skirball Inst Biomol Med, New York, NY 10016 USA
[2] NYU, Sch Med, Dept Cell Biol, New York, NY 10016 USA
关键词
D O I
10.1021/bi010138+
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to1.8 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 muM, 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 mu mol min(-1) mg(-1) at pH 7.0 and 37 degreesC, 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.
引用
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页码:6628 / 6635
页数:8
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