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

Sci Rep. 2019 Feb 4;9(1):1203. doi: 10.1038/s41598-018-37445-2.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Glucose / metabolism
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Membrane Glycoproteins / metabolism
  • Membrane Proteins / metabolism
  • Monosaccharide Transport Proteins / genetics
  • Pichia / genetics
  • Plasmids / genetics
  • Protein Engineering / methods*
  • Recombinant Proteins / genetics
  • Sodium / metabolism
  • Sodium-Glucose Transport Proteins / isolation & purification
  • Sodium-Glucose Transport Proteins / metabolism
  • Sodium-Glucose Transporter 1 / genetics*
  • Sodium-Glucose Transporter 1 / isolation & purification*
  • Sodium-Glucose Transporter 1 / metabolism
  • Workflow

Substances

  • Membrane Glycoproteins
  • Membrane Proteins
  • Monosaccharide Transport Proteins
  • Recombinant Proteins
  • Sodium-Glucose Transport Proteins
  • Sodium-Glucose Transporter 1
  • Green Fluorescent Proteins
  • Sodium
  • Glucose