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.

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: https://theses.gla.ac.uk/id/eprint/1722