Protein Expression and Purification Core Facility
SDS-PAGE analysis after purification of LIF by ion-exchange chromatography.
Our facility produces and purifies proteins from E.coli, insect, mammalian cells and sera using a variety of chromatographic methods. Following each purification, we can perform biophysical analyses to ensure the quality of the purified proteins in terms of correct folding and stability. Our group also develops or evaluates new techniques or advanced protocols for protein production and purification. Most of our efforts are dedicated to developing time-saving solutions for these activities. Moreover, we are keeping stocks of a large number of expression vectors and bacterial strains for the users as well as preparing a collection of frequently used enzymes for general use which helps to considerably reduce the expenses of our users.
Major projects and accomplishments
We have evaluated new variants of our widely used pETM-series expression vectors for E.coli that can now be used for sequence and ligation-independent cloning (SLIC). We have adapted vectors for insect and mammalian cells for the same cloning protocol. Using a single PCR product with the gene of interest, it is possible to integrate the insert into all of the vectors due to the universal overlaps that are present in the linearised vectors and the PCR product. A lethal gene insert in the original template vectors inhibits the growth of false positive colonies, which reduces the number of clones to test for the correct insert. With this new vector set, one can test the expression of a gene in different expression systems in parallel and avoid re-designing of inserts for restriction-based cloning. We have established new vectors for expression of fusion protein based on the small SUMO proteins and their highly specific protease SenP2. In most of our expressions, SUMO-fusion proteins showed high expression yields. In cases of initially insoluble product, we developed a protocol for proteolytic cleavage of the urea-denatured fusion protein with the robust protease under conditions where other proteases show a poor performance. We could obtain pure, untagged proteins that were otherwise difficult to express or purify and could be used, for example, as antigens for immunisation.
Services provided
- Expression and purification of proteins in E.coli, insect and mammalian cells.
- Preparing injection material for immunisations and purification of antibodies from serum and hybridoma supernatants..
- Maintaining collections of expression vectors and bacterial strains.
- Producing frequently used enzymes and protein molecular weight markers for use at EMBL.
- Developing and testing new vectors and protocols.
- Providing access to protocols and vector sequence information on the website.
- Giving scientific and technical advice to internal and external users.
- Caring for protein production and analysis equipment and the cell culture room.
- Providing quality analysis and biophysical characterisation of purified proteins, e.g. analytical ultracentrifugation (AUC) or isothermal titration calorimetry (ITC).
Technology partners
We are open to collaborations with academic or industrial partners to evaluate new products or technologies that could be helpful for improving the service capabilities of our facility. We are frequently approached by company representatives when new products become available, which we test according to the needs of our users.