The Protein Expression and Purification Core Facility produces and purifies proteins both from Escherichia coli and from insect cells. We implement a large array of chromatographic techniques and also provide support for the biophysical characterization of the purified proteins.
The main aim of the facility is to provide assistance and scientific advice concerning all experiments related to protein expression, purification and biophysical characterization. We can perform quality control assays probing the folding state and stability of the recombinant proteins. We are trying to stay up-to-date with the latest scientific developments and trends in the field and test out new products and protocols for our users. Furthermore, we maintain an extensive collection of expression vectors and bacterial strains and we also have some frequently used proteins such as proteases and polymerases readily available. This helps to considerably reduce the expenses of our users.
Major projects and accomplishments
pETM vector set adapted for SLIC
We constructed new variants of our pETM expression vectors for E. coli, which can be used for sequence and ligation-independent cloning (SLIC). We modified other expression vectors for insect and mammalian cells as well for compatibility with 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 ccdB gene in the original template vectors inhibits the growth of false positive colonies. With this new vector set, one can test the expression of a gene in different expression systems in parallel and avoid redesigning of the inserts for restriction-based cloning.
We established a protocol for the expression and purification of fusion proteins based on the small SUMO proteins and their highly specific protease SenP2. In case insoluble products are obtained, SenP2 is capable of proteolytic cleavage of the SUMO-tag under denaturing conditions as well. This allows us to produce pure untagged proteins that are otherwise difficult to express and purify such as antigens for immunizations or cytokines for cell culture.
We are in the process of developing high-throughput screening methods for analytical and preparative scale protein purifications using an automated liquid handling system.
- Expression testing of the protein of interest in various E. coli and insect cell strains and in different culturing conditions;
- Purification of recombinant proteins from E. coli and insect cells;
- Quality control and biophysical characterization of purified proteins (e.g. analytical gel filtration, analytical ultracentrifugation, ITC, CD, thermofluor assays, …);
- Preparation of injection material for immunizations and purifications of antibodies from serum and hybridoma supernatants;
- Production of frequently used proteins (e.g. proteases, polymerases, fluorophores, …) for general use within EMBL;
- Providing scientific and technical advice to internal users at EMBL as well as to external researchers;
- Teaching and training of the users in all aspects of protein expression and purification techniques;
- Maintaining a large collection of expression vectors and bacterial strains;
- Evaluating new protocols and products for our users.
We are open to collaborations with academic and industrial partners to assess new products or technological developments. Furthermore, we have initiated a network of protein facilities across Europe called P4EU (Protein Production and Purification Partnership in Europe (P4EU)) to improve information exchange and the evaluation of novel technologies.