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

The glycerol-3-phosphate (G3P) transporter, GlpT, from Escherichia coli med iates G3P and inorganic phosphate exchange across the bacterial inner membr ane. It possesses 12 transmembrane alpha -helices and is a member of the Ma jor Facilitator Superfamily. Here we report overexpression, purification, a nd characterization of GlpT. Extensive optimization applied to the DNA cons truct and cell culture has led to a protocol yielding similar to1.8 mg of t he transporter protein per liter of E. coli culture. After purification, th is protein binds substrates in detergent solution, as measured by tryptopha n 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 in to proteoliposomes. The phosphate efflux rate of the transporter in the pre sence of G3P is measured to be 29 mu mol min(-1) mg(-1) at pH 7.0 and 37 de greesC, corresponding to 24 mol of phosphate s(-1) (mol of protein)(-1). In addition, the glycerol-3-phosphate transporter is monomeric and stable ove r a wide pH range and in the presence of a variety of detergents. This prep aration of GlpT provides ideal material for biochemical, biophysical, and s tructural studies of the glycerol-3-phosphate transporter.