Structural biology workflow for the expression and characterization of functional human sodium glucose transporter type 1 in Pichia pastoris

Albert Suades, Antonio Alcaraz, Esteban Cruz, Elena Álvarez-Marimon, Julian P. Whitelegge, Joan Manyosa, Josep Cladera, Alex Perálvarez-Marín

Research output: Contribution to journalArticleResearch

2 Citations (Scopus)

Abstract

© 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.
Original languageEnglish
Article number1203
JournalScientific Reports
Volume9
DOIs
Publication statusPublished - 1 Dec 2019

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