For my UROP placement I have been working between two life science labs in the Harwell Science and Innovation campus near Oxford; Membrane Protein Lab (MPL) and The Research Complex, along with a PhD student, Frank Atanu, supervised by Dr. Kim Watson. The overall project aim is to elucidate the protein structure of Chlamydia Pneumoniae’s major outer membrane protein (MOMP) in addition to analysing thermal stabililty, UV degradation, and ligand binding, all through CD spectroscopy, using the beamline at Diamond Light Source.
MOMP is referred to as an immune response protein, due to it’s anti-atherosclerotic effects achieved by reducing MHC-II protein activity. Due to the complications involved in culturing C. pneumoniae cells, MOMP is encoded into a gene transcript which is inserted into E. coli C41 cells downstream of the lac operon expression system. Upon induction at the critical growth stage with IPTG, a synthetic analogue of lactose, the lac operon is activated resulting in transcription and thus MOMP production. Approximately 20 hours post induction the membranes are harvested through ultracentrifugation and the proteins subsequently purified via affinity chromatography. Following the running of a Coomassie gel to determine the presence (or absence!) of the protein of interest the protein is then concentrated to a volume of around 500µl via centrifugation with a 30 kDa centrifugal filter. A machine, the äkta purifier, uses fast protein liquid chromatography (FPLC) to separate the protein, which if pure will be displayed as a single peak. Regarding this information the protein can then be collected from the sample wells where the peak was detected, resulting in a pure protein sample, which could then be analysed for folding properties and secondary structures by circular dichroism (CD) spectophotometry on the beamline at Diamond Light Source (DLS). I also had the opportunity to create crystallization plates for both MOMP and the four MHC-II peptides, using the mosquito and Cartesian machines respectively. These 96 well plates are then routinely photographed and stored online to allow for visual analysis of crystal formation.
The final stretch of my placement involved a weekend stay (Friday-Monday) at DLS, with accommodation provided on site, to spend some intensive time analyzing MOMP and MHC-II with CD spectroscopy. To make full use of the precious time we were up all hours collecting data, as following our 72 hour slot was another researcher’s project. It has been very advantageous for the project to have the opportunity to use the equipment available at DLS, which enabled us to collect a large amount of data on UV degradation, thermal stability and MOMP-ligand interactions which will no doubt provide valuable insights in the stability of the protein structure as well as potential binding pockets and interactions.
Despite the long commuting hours from my house in Reading to the Harwell campus, which involved catching two buses and a train, I feel very privileged to have had the opportunity to work in labs with such facilities amongst some fellow students and researchers that have made me feel more than welcome. For anyone uncertain whether a role in scientific research is a career they’d like to pursue, I highly recommend you take this brilliant opportunity, as not only does it provide you with career experience but also helps to develop your lab skills, giving you extra confidence ahead of your third year research project.
Amy Danson
Bsc Biomedical Sciences UROP 2013