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

FEBS Letters

Wright, Ernest M¹  Panayotova-Heiermann, Mariana¹  Hirayama, Bruce A¹  Leung, Daisy W¹ 

[1] Department of Physiology, UCLA Medical Center, CHS Box 951751, Los Angeles, CA 90095-1751, USA

关键词: Human SGLT1;    GST fusion protein;    Purification;    Reconstitution;    SGLT1;    high affinity Na+-dependent cotransporter for D-glucose;    αMDG;    α-methyl-D-glucopyranoside;    SDS-PAGE;    sodium dodecylsulfate-polyacrylamide gel electrophoresis;    IPTG;    isopropyl 1-thio-β;    D-galactopyranoside;   

DOI  :  10.1016/S0014-5793(99)01292-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown