The Membrane Protein Laboratory (MPL) at the Research Complex Harwell is a research and training user facility open to scientists from laboratories anywhere in the world that are interested in the Structural Biology of Membrane Proteins. The laboratory combines recently developed high throughput technologies for membrane protein production with sample preparation for crystallisation and cryo-electron microscopy. As a Wellcome funded support facility within Diamond Light Source we are uniquely placed to utilise the latest state-of-the-art developments in X-ray diffraction data collection and cryo-electron microscopy.
Membrane proteins are some of the most difficult proteins to study in structural biology and require a significant commitment in terms of time and financial resources. The ability to quickly identify the most suitable expression system and purification conditions using minimal material significantly reduces the initial outlay of research time and financial resources rapidly advances the early stages of a project. At the MPL we can support visiting scientists to rapidly clone and screen between 48 and 96 constructs simultaneously in either E. coli, insect or mammalian cell culture on a small scale (1-3ml of cell-culture per condition). By utilising commonly used purification tags (e.g. His, FLAG, Twin-Strep, GST) and reporter proteins (e.g. GFP, RFP, YFP) we can quickly identify the most suitable expression and purification conditions (e.g. incubation time and temperature, solvent composition; detergents, pH, salts and other additives). SDS-PAGE gels and fluorescence size exclusion (F-SEC) are used to judge success. Fractions from F-SEC can be collected and be assessed by negative stain cryo-EM depending on purification yield. We are grateful for the support of Instruct-ULTRA for providing support towards the development of our small scale workflows to support membrane protein expression and purification screening.
High quality membrane protein samples are essential for structural biology. Our protein production platform can support visiting scientists to clone, express and purify 48-96 membrane protein constructs on a small scale. We provide the facilities to utilise protein expression in E.coli, yeast, insect and mammalian cells. This is coupled to a high-throughput screening platform to rapidly identify the most suitable conditions for membrane protein purification. Our expression platform has recently been enhanced through the work of an Instruct-ULTRA post-doctoral research associate who developed new protocols and enhanced workflows for membrane protein production in mammalian cells.
Visiting scientists can enter the workflow at any point providing protein constructs are compatible with our screening platform. There is no requirement for fluorescent tags but this will limit some of the optimisation that can be done during expression screening stage. The membrane protein expression screening platform allows an initial purification on a small scale while membrane protein purification and formulation allows scientists to screen a wider range of conditions including but not limited to detergents, amphipols, membrane scaffold proteins (MSPs), SMALPS, lipids, pH and salts. Please contact the facility for more information.