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

Abstract

A truncated human Na + /glucose cotransporter (C 5 , residues 407^664) was expressed and purified from Escherichia coli 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 1 mg of purified GST-C 5 was obtained from 1 l 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 [ 3 H]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.z 1999 Federation of European Biochemical Societies.

Three Surface Subdomains Form the Vestibule of the Na+/Glucose Cotransporter SGLT1

“…Therefore, we hypothesized that this region in subdomain II either lies close to the sugar-binding site, which appears only in the presence of Na ϩ , or represents part of the initial binding pocket (vestibule), which subsequently transfers glucose to the translocation pathway. Several groups have proposed that the glucose translocation region lies between helices 10 -13 of SGLT1, however, with low affinity to D-glucose and lost sensitivity to phlorizin (10,13). In this study, we suggest that the residues between aa 339 and 356 located between helix 8 and 9 also contribute to efficient high affinity D-glucose transport.…”

“…Many researchers have attempted to localize a substrate translocation pathway in SGLT1. Studies on chimera proteins of SGLT1/SGLT2 and a truncated protein (residues 407-648) showed that residues from 381-662 of SGLT1 are important for sugar transport, however, with less sugar specificity and lower sensitivity to the competitive inhibitor phlorizin (10,13). Also residues 457, 468, and 499 have been shown to play an important role in controlling the sugar binding of SGLT1 (14,15).…”

“…So far, there is evidence showing one disulfide bridge between residues Cys 255 and Cys 511 in human SGLT1 (23). In rabbit SGLT1 there are three large extramembranous loops or surface subdomains (loop 6 -7, loop 8 -9, and loop [13][14] that contain cysteine residues and therefore are potential sites for bringing the transmembrane helices 4 and 5 close to the transmembrane helices 10 -13. Thereby, a vestibule containing a glucose recognition site might be formed similar to the results from the ␥-aminobutyric acid transporters, showing that three surface loops form a pocket in which the substrate initially binds to the transporters (24).…”

Sequence Analysis of a 101-Kilobase Plasmid Required for Agar Degradation by aMicroscillaIsolate

“…It is possible that pSD15 encodes the symporter in three parts, which are assembled together to form a functional unit. Na ϩ /glucose transporters have been shown to utilize a Na ϩ electrochemical gradient to drive sugar transport in mammalian cells (33), but except for the SglS transporter from V. parahaemolyticus, this is unusual in bacteria (37). A conserved amino acid sequence (G. .…”

Employing Escherichia coli to functionally express, purify, and characterize a human transporter

“…Here, we report the employment of Escherichia coli for the expression, purification, and reconstitution of a fully functional recombinant human sodium͞glucose transporter (hSGLT1)-a central protein for the homeostasis of glucose, salt, and water (12)(13)(14). Our strategy is based in part on our group's previous success in purifying functional transporters from bacteria (15,16).…”