Breen, Conor and Martin, Darren S. and Ma, Hui and McQuaid, Kate and O'Kennedy, Richard and Findlay, John B. C. (2015) Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination. PLoS ONE, 10 (3). e0122293. ISSN 1932-6203

Preview

Download (792kB) | Preview

Abstract

STRA6 is a plasma membrane protein that mediates the transport of vitamin A, or retinol, from plasma retinol binding protein (RBP) into the cell. Mutations in human STRA6 are associated with Matthew-Wood syndrome, which is characterized by severe developmental defects. Despite the obvious importance of this protein to human health, little is known about its structure and mechanism of action. To overcome the difficulties frequently encountered with the production of membrane proteins for structural determination, STRA6 has been expressed in Pichia pastoris as a fusion to green fluorescent protein (GFP), a strategy which has been a critical first step in solving the crystal structures of several membrane proteins. STRA6-GFP was correctly targeted to the cell surface where it bound RBP. Here we report the large-scale expression, purification and characterisation of STRA6GFP. One litre of culture, corresponding to 175 g cells, yielded about 1.5 mg of pure protein. The interaction between purified STRA6 and its ligand RBP was studied by surface plasmon resonance-based binding analysis. The interaction between STRA6 and RBP was not retinol-dependent and the binding data were consistent with a transient interaction of 1 mole RBP/mole STRA6.

Item Type:	Article
Additional Information:	© 2015 Breen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Breen CJ, Martin DS, Ma H, McQuaid K, O’Kennedy R, Findlay JBC (2015) Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination. PLoS ONE 10(3): e0122293. doi:10.1371/journal.pone.0122293
Keywords:	Production; Vitamin A; Retinol; Transporter; Receptor; STRA6; Structure Determination; plasma retinol binding protein; RBP; Matthew-Wood syndrome;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	6855
Identification Number:	https://doi.org/10.1371/journal.pone.0122293
Depositing User:	Professor John Findlay
Date Deposited:	19 Jan 2016 14:51
Journal or Publication Title:	PLoS ONE
Publisher:	Public Library of Science
Refereed:	Yes
Funders:	Science Foundation Ireland (SFI)
URI:	http://journals.plos.org/plosone
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

Repository Staff Only(login required)

Item control page

Downloads

Origin of downloads