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Development of a high throughput screen for the expression of membrane proteins and their purification and crystallisation

Kroner, Frank (2010) Development of a high throughput screen for the expression of membrane proteins and their purification and crystallisation. PhD thesis, University of Glasgow.

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Abstract

Every biological cell is surrounded by a membrane, which functions as a barrier to the environment and as a support matrix for membrane proteins. Membrane proteins facilitate the transport of manifold substrates across the membrane and are involved in fundamental cellular processes, such as signalling or energy generation to name a few. The key to the function of membrane proteins lies in their three dimensional structure, which can be determined by single crystal X-ray crystallography. However, membrane proteins are one of the most difficult protein classes to work with, which is reflected by the small number of available membrane protein structures. Protein crystallography requires milligram amounts of pure protein, which has to be expressed and purified to monodispersity to allow crystallisation. As membrane proteins have to be inserted into the membrane, recombinant expression yields are often low. In order to obtain enough protein for purification and crystallisation studies, the expression of membrane proteins requires screening for the best expression conditions. Purification of membrane proteins requires, due to their amphipathic character, the use of detergents to solubilise the membrane protein. The optimal combination of detergent and membrane protein is crucial for stability in aqueous solution in order to allow purification to monodispersity. Furthermore, the detergent has a high influence on the crystallisation of membrane proteins. An approach to overcome the challenges of membrane protein structural biology is to work in a high throughput (HTP) manner to increase the chances of success. The aim is to find the most promising targets out of a library of membrane proteins and in the presented work a small-scale HTP expression screen was developed in order to find the optimal expression conditions for each membrane protein from a target library of 12 E. coli inner membrane proteins. The targets were then expressed in the determined optimal conditions in sufficient amounts to allow purification. All membrane proteins were subjected to a purification pipeline, which employed a subset of parameters, that have proved to be the most successful to date in membrane protein purification for structural studies. Five membrane proteins were purified to monodispersity and were submitted to crystallisation trials. Crystals of two targets were obtained, which diffracted to 7 Å and 15 Å. Furthermore, the data collected on the expression and purification behaviour of the 12 membrane proteins, will help to optimise the starting parameters for the screening of future targets.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: membrane proteins, high throughput, expression screen, detergent optimisation, protein crystallisation, GFP.
Subjects: Q Science > QD Chemistry
Colleges/Schools: College of Science and Engineering > School of Chemistry
Supervisor's Name: Isaacs, Prof. Neil W.
Date of Award: 2010
Depositing User: Mr Frank/ F Kroner
Unique ID: glathesis:2010-1722
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 21 Apr 2010
Last Modified: 10 Dec 2012 13:45
URI: http://theses.gla.ac.uk/id/eprint/1722

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