SDS-PAGE analysis after purification of LIF by ion-exchange chromatography

The facility produces and purifies proteins from E. coli, insect, mammalian cells and sera, using a variety of chromatographic methods and provides support for biophysical characterisation of purified proteins.

Following each purification, we can perform biophysical analyses to ensure the quality of the purified sample in terms of correct folding and stability. Our facility also develops or evaluates new techniques and advanced protocols for protein production and purification and there is significant focus on 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 proteins for general use, which helps to considerably reduce the expenses of our users.

Major projects and accomplishments

We have evaluated new variants of our 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 ccdB gene 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 the redesigning of inserts for restriction-based cloning.

We have established a generic protocol 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 insoluble product, we developed a protocol for proteolytic cleavage of the ureadenatured fusion protein, with the robust protease under conditions where other proteases show a poor performance. We can obtain pure, untagged proteins that are otherwise difficult to express or purify which include cytokines for cell culture or antigens for immunisation.

Services provided

  • Expression and purification: proteins in E. coli, insect and mammalian cells.
  • Scientific and technical advice to users at EMBL and external researchers.
  • Preparing injection material for immunisations and purification of antibodies from serum and hybridoma supernatants.
  • Providing quality analysis and biophysical characterisation of purified proteins (ITC, analytical ultracentrifugation, CD).
  • Maintaining collections of expression vectors and bacterial strains.
  • Producing frequently used enzymes and protein molecular weight markers for general use within EMBL.
  • Developing and testing new vectors and protocols.
  • Access to protocols and vector sequence information on the website.
  • Caring for equipment for protein production and analysis.

    Technology partners

    We are open to collaborations with academic or industrial partners to evaluate 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 evaluation of new technologies.