Structural biology workflow for the expression and characterization of functional human sodium glucose transporter type 1 in Pichia pastoris
Suades, Albert (Stockholm University. Department of Biochemistry and Biophysics)
Alcaraz, Antonio (Universitat Jaume I. Departament de Física)
Cruz, Esteban (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Álvarez Marimon, Elena (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Whitelegge, Julian P. (David Geffen School of Medicine)
Manyosa Ribatallada, Joan (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Cladera i Cerdà, Josep (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Peralvarez-Marin, Alex (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)

Date:	2019
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.
Grants:	European Commission 237120 Ministerio de Ciencia e Innovación BFU2012-40137-C02-01 Ministerio de Ciencia e Innovación SAF2010-21385
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Published in:	Scientific reports, Vol. 9 (February 2019) , art. 1203, ISSN 2045-2322

DOI: 10.1038/s41598-018-37445-2
PMID: 30718602

11 p, 2.1 MB

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