comunitat-uji-handle:10234/9
comunitat-uji-handle2:10234/2507
comunitat-uji-handle3:10234/6973
comunitat-uji-handle4:
INVESTIGACION
Metadata
Title
Structural biology workflow for the expression and characterization of functional human sodium glucose transporter type 1 in Pichia pastoris
Date
2019
Publisher
Nature Research
ISSN
2045-2322
Bibliographic citation
Suades, Albert, et al. "Structural biology workflow for the expression and characterization of functional human sodium glucose transporter type 1 in Pichia pastoris." Scientific reports, 2019, vol. 9, núm. 1
Type
info:eu-repo/semantics/article
Version
info:eu-repo/semantics/publishedVersion
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 ... [+]
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. [-]
Is part of
Scientific reports, 2019, vol. 9, núm. 1
Investigation project
This work was supported by the MICINN grants BFU2012-40137-C02-01 to J.C. and SAF2010-21385 to A.P.-M., and the NIH grant NIH P30 DK063491 to J.P.W.; A.P.-M. was a recipient of a Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme (PIOF-GA-2009-237120 to A.P.-M.) and a Universitat Autònoma de Barcelona-Programa Banco de Santander Fellowship. A.S. was a recipient of a Ph.D. fellowship and a travel bursary from Universitat Autònoma de Barcelona
Rights
info:eu-repo/semantics/openAccess
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