Research objectives and content
The objective of my research project is the directed evolution of an alpha/beta-barrel protein to create a new catalytic activity on the existing scaffold. The aim then is to characterise both the intermediate and the final proteins identified during the directed evolution process using both functional and structural probes. This will give a unique insight at a molecular level into the way a protein evolves in response to a selective pressure, and into how the changes in function correlate with changes in structure.
The project will involve creation and expression of libraries of the alpha/beta-barrel protein indoleglycerolphosphate synthase in appropriate strains of E. coli. Mutation of the cloned wild-type gene in both a targeted and random manner will be followed by selection for the ability to complement suitable auxotrophic strains of E. coli. Iteration of this process in combination with 'DNA shuffling' will be used to identify new variants with improved activity. Selected proteins isolated will be over-expressed, purified and used for crystallisation experiments. If crystals are obtained, I will undertake to solve the structures by x-ray crystallography; this should be greatly simplified by use of molecular replacement methods since the wild-type structure has been solved previously at high resolution.
Training content (objective, benefit and expected impact)
My project will be part of a research programme for a postgraduate degree from Cambridge University, and it will provide advanced scientific training for a career in molecular and structural biology. My project will be co-supervised by two members of the academic staff in the Biochemistry Department and I will benefit greatly from the environment since I will join two experienced research groups. These groups will between them provide access to facilities for molecular biology, display technologies, enzymology, preparative biochemistry, computation and x-ray data collection, and I will gain experience in each of these. The research groups will also provide specialised reagents needed for my project. Links with industry / industrial relevance (22)
At the onset, there are no industrial links, but the results will quite possibly have application in a rational and generic approach to the design of novel enzymes. This would be of potential significance in both the pharmaceuticals industry and directly in medicine. Proteins with novel catalytic activities might be used in the synthesis of new drugs or, in vivo, for intracellular hydrolysis of prodrugs, thus increasing the specificity of such therapy. In an extracellular environment, enzymes with modified activities could be used in diagnostics and even to intervene with accessible disease states.