The over-expression, purification and crystallisation of the alternative oxidase

Elliott, Catherine Rebecca (2013) The over-expression, purification and crystallisation of the alternative oxidase. Doctoral thesis (PhD), University of Sussex.

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The alternative oxidase (AOX) is an integral monotopic membrane protein which
branches from respiratory chain at the point of the Q-pool in the mitochondria of all
flowers, some fungi, and some protists such as the human parasite Trypanosoma brucei.
The aim of this project is threefold: to establish an over-expression and purification
protocol for recombinant Sauromatum guttatum alternative oxidase (SgrAOX); to use
expressed SgrAOX for structural analysis such as crystallography; and finally to use in
silico methods to model the alternative oxidase protein. Of these three, only the first and
last have been attempted previously, with varying success. The second, namely
structural analysis, has never been attempted with SgrAOX.
In order to achieve the aims of this project, primarily laboratory-based protein
production were used, in conjunction with downstream analysis using structural biology
techniques. The in silico modelling was carried out using a wide range of algorithms
freely available on the World Wide Web.
Results of this project are: the determination of an over-expression system and
purification protocols in two E.coli strains, producing enough protein to use for the
second objective detailed above. While no crystal structure has been obtained,
significant steps toward identifying a protocol for rAOX crystallisation have been made.
Results from structural analysis support modelling predictions and give novel insights
into the thermostability of the protein. New and detailed homology models have been
created and critically evaluated, with a very recent crystal structure from our
collaborators providing a unique set of data for model evaluation.
The outcome of this project has contributed towards the determination of conditions
under which SgrAOX protein may form crystals, and therefore bringing the acquisition
of a SgrAOX protein structure closer.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Depositing User: Library Cataloguing
Date Deposited: 06 Dec 2013 14:46
Last Modified: 17 Sep 2015 13:49

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