Title of project: Development of novel membrane protein purification technology for
structural studies
Director of Studies: Dr Vincent Postis
Second Supervisor: Dr Tara Sabir
Overview of project
Unlike soluble protein, membrane protein structural studies have been hindered for many years due
to various limitations in protein expression and purification. Protein expression issues have been
addressed; however the purification of membrane proteins is still problematic. The main issue is the
use of detergents for protein solubilisation and downstream processes such as reconstitution and
biophysical characterisation. Not only is the right detergent formulation required to keep the protein in
a native functional state, but the detergent must be easy to remove to limit interference with protein
assays.
The use of protein scaffolds has revolutionised membrane protein purification. Two types of scaffold
are currently employed to maintain integrity of the membrane protein in lipoparticule: protein scaffold
(nanodiscs) and polymer scaffold (SMALPs). Prior to reconstitution in nanodisc, membrane proteins
require to be solubilised by detergents. SMALP are on the other hand employed as molecular cutters
allowing co-purification of the membrane proteins with their natural lipidic environment. Each
approach has its pros and cons, the use nanodiscs or SMALPs is restricted by the type of protein and
the nature of the downstream biophysical assays.
In order to understand the advantages and limitations of each technique, this project aims at studying
two model proteins and characterise them through a range of biophysical techniques such as Mass
-barrel protein, OmpX,
have been chosen for these studies. Both membrane proteins are well characterised and expression
conditions are already established. AcrB is involved in antibiotic resistance while OmpX is a bacterial
porin. Both proteins will be solubilised in classical conditions (detergent) as a control. The proteins will
be reconstituted in nanodiscs and isolated in SMALP. Subsequently the particles will be tested for
structural studies. The protein expression plasmids required for this project are ready for use and
technical training will be provided by Dr. Postis who has extensive expertise in membrane protein
purification
Link to Faculty Research Themes
School of Rehabilitation and Health Sciences and University of Leeds. 5A (Biological Sciences).
Outline of project including proposed timescales
This project will provide training in biochemistry and biophysical assays. Papers published from this
study will be submitted to high impact journals, such as JACS (impact factor 11.4) and Biochemical
Journal (impact factor 4.8). This work outcome will be used as supporting data for a grant application
to the BBSRC. At the end of this PhD program, the successful candidate will have acquired training in
cutting edge methodologies, which are in high demand for future employment.
Further information
To apply you must be eligible for NHS Continuing Professional Development (CPD) funding and have
the support of your line manager in writing. General enquiries should be directed by email to the
Faculty Research Director r.hogston@leedsbeckett.ac.uk to discuss the project further please contact
the Director of Studies V.L.Postis@leedsbeckett.ac.uk
Applications should be made on line here
http://www.leedsbeckett.ac.uk/research/research-degrees/research-studentships-andfees-only-bursaries/