TY - JOUR
T1 - Structural biology workflow for the expression and characterization of functional human sodium glucose transporter type 1 in Pichia pastoris
AU - Suades, Albert
AU - Alcaraz, Antonio
AU - Cruz, Esteban
AU - Álvarez-Marimon, Elena
AU - Whitelegge, Julian P.
AU - Manyosa, Joan
AU - Cladera, Josep
AU - Perálvarez-Marín, Alex
PY - 2019/12/1
Y1 - 2019/12/1
N2 - © 2019, The Author(s). Heterologous expression of human membrane proteins is a challenge in structural biology towards drug discovery. Here we report a complete expression and purification process of a functional human sodium/D-glucose co-transporter 1 (hSGLT1) in Pichia pastoris as representative example of a useful strategy for any human membrane protein. hSGLT1 gene was cloned in two different plasmids to develop parallel strategies: one which includes green fluorescent protein fusion for screening optimal conditions, and another for large scale protein production for structural biology and biophysics studies. Our strategy yields at least 1 mg of monodisperse purified recombinant hSGLT1 per liter of culture, which can be characterized by circular dichroism and infrared spectroscopy as an alpha-helical fold protein. This purified hSGLT1 transports co-substrates (Na+ and glucose) and it is inhibited by phlorizin in electrophysiological experiments performed in planar lipid membranes.
AB - © 2019, The Author(s). Heterologous expression of human membrane proteins is a challenge in structural biology towards drug discovery. Here we report a complete expression and purification process of a functional human sodium/D-glucose co-transporter 1 (hSGLT1) in Pichia pastoris as representative example of a useful strategy for any human membrane protein. hSGLT1 gene was cloned in two different plasmids to develop parallel strategies: one which includes green fluorescent protein fusion for screening optimal conditions, and another for large scale protein production for structural biology and biophysics studies. Our strategy yields at least 1 mg of monodisperse purified recombinant hSGLT1 per liter of culture, which can be characterized by circular dichroism and infrared spectroscopy as an alpha-helical fold protein. This purified hSGLT1 transports co-substrates (Na+ and glucose) and it is inhibited by phlorizin in electrophysiological experiments performed in planar lipid membranes.
U2 - 10.1038/s41598-018-37445-2
DO - 10.1038/s41598-018-37445-2
M3 - Article
C2 - 30718602
VL - 9
M1 - 1203
ER -