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Over-expression and biophysical characterisation of membrane proteins solubilised in a styrene maleic acid polymer

Lin, Yu-pin (2011)
Ph.D. thesis, University of Birmingham.

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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.

Type of Work:Ph.D. thesis.
Supervisor(s):Dafforn, Timothy
School/Faculty:Colleges (2008 onwards) > College of Life & Environmental Sciences
Department:School of Biosciences
Subjects:QR Microbiology
Institution:University of Birmingham
ID Code:1738
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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