title: Production of functional human vitamin A transporter/RBP receptor (STRA6) for structure determination.
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 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.
authors: Department of Biology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland.
authors: Conor J
authors: CJ
authors: Breen
authors: Department of Biology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland.
authors: Darren S
authors: DS
authors: Martin
authors: National Centre for Sensor Research, Biomedical Diagnostics Institute, Dublin City University, Dublin, Ireland.
authors: Hui
authors: H
authors: Ma
authors: Department of Biology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland.
authors: Kate
authors: K
authors: McQuaid
authors: National Centre for Sensor Research, Biomedical Diagnostics Institute, Dublin City University, Dublin, Ireland.
authors: Richard
authors: R
authors: O'Kennedy
authors: Department of Biology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland.
authors: John B C
authors: JB
authors: Findlay
mesh_terms: D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054839:Retinol-Binding Proteins, Plasma / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D054839:Retinol-Binding Proteins, Plasma; C519685:STRA6 protein, human; D049452:Green Fluorescent Proteins