Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination
| Autor: | Kate McQuaid, Hui Ma, John B. C. Findlay, Conor J Breen, Richard O'Kennedy, Darren S. D. Martin |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
endocrine system
Recombinant Fusion Proteins Green Fluorescent Proteins lcsh:Medicine Plasma protein binding Biology Pichia Green fluorescent protein Pichia pastoris Humans Receptor lcsh:Science Multidisciplinary lcsh:R Lipid bilayer fusion Membrane Proteins Surface Plasmon Resonance Ligand (biochemistry) biology.organism_classification Retinol binding protein Membrane protein Biochemistry lcsh:Q Retinol-Binding Proteins Plasma Protein Binding Research Article |
| Zdroj: | PLoS ONE, Vol 10, Iss 3, p e0122293 (2015) PLoS ONE |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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