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

A truncated human Na⁺/glucose cotransporter (C₅, residues 407-664) was expressed and purified from Escherichia coli using a GST fusion vector and glutathione affinity chromatography. The truncated transporter (C₅) was cleaved from GST-C₅ by Factor Xa proteolysis and purified by gel filtration chromatography. Up to 1 mg of purified GST-C₅ was obtained from 1 l bacterial culture. Reconstitution of both GST-C₅ and C₅ proteins into lipid vesicles resulted in 2.5-fold higher initial uptake rates of [³H]D-glucose into C₅-proteoliposomes than into liposomes. Transport was stereospecific, saturable, and inhibited by phloretin. These properties are similar to those obtained for C₅ in Xenopus laevis oocytes, and provide additional evidence that the five C-terminal transmembrane helices in SGLT1 form the sugar translocation pathway. Copyright (C) 1999 Federation of European Biochemical Societies.