Production of functional human vitamin A transporter/RBP receptor (STRA6) for structure determination

• Published in: PloS one Vol. 10; no. 3; p. e0122293
• Main Authors: Breen, Conor J, Martin, Darren S, Ma, Hui, McQuaid, Kate, O'Kennedy, Richard, Findlay, John B C
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 27-03-2015; Public Library of Science (PLoS)
• Subjects: Biology; Cell culture; Cell surface; Cloning; Crystal defects; Crystal structure; Crystallization; Fluorescence; Fusion protein; Green fluorescent protein; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Humans; Insulin resistance; Ligands; Localization; Membrane proteins; Membrane Proteins - biosynthesis; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Microscopy; Mutagenesis; Mutation; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Protein Binding; Protein transport; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Retinol-Binding Proteins, Plasma - metabolism; Signal transduction; Surface Plasmon Resonance; Vitamin A; Wood; Yeast; Ireland
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0122293

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 STRA6-GFP. 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.