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

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...

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Bibliographic Details
Published in:PloS one 2015-03, Vol.10 (3), p.e0122293-e0122293
Main Authors: Breen, Conor J, Martin, Darren S, Ma, Hui, McQuaid, Kate, O'Kennedy, Richard, Findlay, John B C
Format: Article
Language:English
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
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0122293