Lin, Yu-pin
(2011).
Over-expression and biophysical characterisation of membrane proteins solubilised in a styrene maleic acid polymer.
University of Birmingham.
Ph.D.
Abstract
Membrane proteins are the gateways into cells and therefore a critical part of pharmaceutical research. Membrane proteins, in fact, have been shown to be the target of over 50% of all medicinal drugs. The development of drugs that acts on membrane proteins, however, has been limited by the lack of high resolution structural data. This lack of information results from a lack of a generic method to extract active membrane proteins from the cell membrane. We have developed a novel styrene maleic acid (SMA) / lipid particle system termed SMALP that preserves the structural and functional integrity of membrane proteins. We have optimised this system and used it to solubilise two membrane proteins with different architectures and functions. The first, human adenosine 2a receptor (A2aR), is a G-protein coupled receptor, a 7 transmembrane helix protein that is involved in cell signalling, whereas the second, FtsZ-interacting protein A (ZipA), is a protein involved in bacterial cell division that contains a single transmembrane domain. In this project, standardised purification protocols were developed that produced pure proteins without the need to add any detergents. The subsequent biophysical characterisation that included circular dichroism, mass spectrometry and sedimentation velocity analytical ultracentrifugation indicated that these purified SMALP solubilised proteins were natively folded once encapsulated. The activities of both proteins were then tested and shown to be close to that expected for the intact proteins in the cell membrane. Taken together these data suggest that the SMALP system offers the solution to membrane protein purification into the future.
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