Purification and functional reconstitution of a truncated human Na+/glucose cotransporter (SGLT1) expressed in E-coli

A truncated human Na+/glucose cotransporter (C-5, residues 407-664) was expressed and purified from Escherichia call using a GST fusion vector and glutathione affinity chromatography, The truncated transporter (C-5) was cleaved from GST-C-5 by Factor Xa proteolysis and purified by gel filtration chromatography, Up to I mg of purified GST-C-5 was obtained from 1 I bacterial culture; Reconstitution of both GST-C-5 and C-5 proteins into lipid vesicles resulted in 2,5-fold higher initial uptake rates of [H-3]D-glucose into C-5-proteoliposomes than into liposomes, Transport was stereospecific, saturable, and inhibited by phloretin, These properties are similar to those obtained for C-5 in Xenopus laevis oocytes, and provide additional evidence that the five C-terminal transmembrane helices in SGLT1 form the sugar translocation pathway. (C) 1999 Federation of European Biochemical Societies.