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Updated: Jan 29, 2026

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies
Published on: January 23, 2019
Structural biology workflow for the expression and characterization of functional human sodium glucose transporter
Albert Suades1,2, Antonio Alcaraz3, Esteban Cruz1
1Biophysics Unit, Department of Biochemistry and Molecular Biology, School of Medicine, Universitat Autònoma de Barcelona, 08193, Cerdanyola del Vallés, Catalonia, Spain.
Abstract:
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.
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