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.
Original language | English |
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Article number | 1203 |
Pages (from-to) | 1203 |
Number of pages | 11 |
Journal | Scientific Reports |
Volume | 9 |
Issue number | 1 |
DOIs | |
Publication status | Published - 1 Dec 2019 |
Keywords
- COTRANSPORTER SGLT1
- EUKARYOTIC MEMBRANE-PROTEINS
- INSIGHTS
- LOOP-13
- MECHANISM
- OPTIMIZATION
- OVEREXPRESSION
- PERMEASE
- PURIFICATION
- RECOGNITION