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

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.

Hydroxyapatite Formation on Self-Assembling Peptides with Differing Secondary Structures and Their Selective Adsorption for Proteins

“…The −C=O stretching vibration at 1600–1700 cm −1 for amide I could be assessed as the peptide structure. Among these, we focused on two main peaks at around 1650 and 1630 cm −1 , attributed to α-helix and β-sheet structure [48,50]. The LELL–HAp (1 and 3 mg) spectra had peaks corresponding to HAp; the PO 4 3− bending vibration (O–P–O) at 560 and 600 cm −1 and the peaks at around 961 and 1024 cm −1 originated from bending modes of the P–O bond in PO 4 3− .…”

TRPV2: A Key Player in Myelination Disorders of the Central Nervous System

“…Rat TRPV2 FL tagged with a C-terminal GFP and a 8xHis tag was overexpressed in yeast Pichia pastoris following our lab protocol as described in Suades et al [ 52 ]. Yeast carrying the construct of interest was grown in YPD Zeocin at 30 °C.…”

Heterologous (Over) Expression of Human SoLute Carrier (SLC) in Yeast: A Well-Recognized Tool for Human Transporter Function/Structure Studies

“…This process, named post-transformational vector amplification (PTVA), resulted in cells containing multiple head-to-tail copies of the vector integrated at a single locus in the genome which would allow a proportional increase in recombinant protein [ 167 ]. However, there is not always a linear correlation between copy number and protein titer or quality [ 168 ]; thus the overall functional expression levels need to be experimentally assessed [ 27 ]. Expression screening allows identification of the best recombinant strain in terms of transporter expression.…”