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 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.
Panayotova-Heiermann
M
M
Department of Physiology, UCLA Medical Center, CHS Box 951751, Los Angeles, CA 90095-1751, USA. mheiermann@mednet.ucla.edu
Leung
D W
DW
Hirayama
B A
BA
Wright
E M
EM
D004926:Escherichia coli / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1; D013997:Time Factors
D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; D005982:Glutathione Transferase; D005947:Glucose