Note: The facility provides vectors, reagents and protocols, however the actual cloning has to be performed by the applicant.
We have three different expression systems available for recombinant protein expression:
- Bacteria (E. coli): various strains, depending on protein to be expressed. Note: Only Phage –resistant bacterial strains are allowed in our lab to prevent phage infections. Proteins can be expressed up to 8 liter culture per prep.
- Insect cell (Sf9): Baculovirus constructs are prepared to infect Sf9 insect cells for expression of both intracellular and secreted proteins. Up to 4 liter cell culture can be prepared per prep.
- Mammalian cells (HEK293 and derivatives). We use this system mainly for expression of extracellular proteins (though intracellular protein expression is possible). Both transient expression and expression from stable cell lines are feasible in plates and roller bottles .
The facility has ample experience with production of monoclonal antibodies from hybridoma cell lines. Cells are cultured in roller bottles and - depending on expression levels – several milligrams of antibody can be produced and purified. Hybridoma cell lines have to be provided by the applicant (external projects) or should be accessible from the NKI hybridoma bank (For NKI researchers only!).
The facility is well-equipped for purification of (recombinant) proteins via multi-step protein purification strategies, including affinity-, ion exchange- and size exclusion chromatography steps. If possible, affinity tags can be cleaved off from the protein by specific proteases during purification. Typical purification yields are between 0.5 and 30 mg of protein.
As a standard quality control procedure, purified proteins are subjected to size exclusion chromatography (size/aggregation analysis); checked by SDS-PAGE analysis (purity) and quantified by absorbance measurements at 280 nm (NanoDrop).
In addition, the stoichiometry and aggregation status of purified protein (-complexes) can be assessed by SEC-MALS. The (thermal) stability of proteins can be examined by nanoDSF where intrinsic tryptophan fluorescence (protein unfolding) and light scattering (protein aggregation) are recorded. The stability can be checked in solutions with different compositions (pH, salt, additive, …) to identify the optimal purification- and/or storage buffer.
The facility can also assists with design and execution of enzyme-kinetic assays, including simulation and fitting of data using the KinTek software package.
The facility houses a selection of instruments for exploring molecular interactions of proteins (see Equipment and Technologies page).
Upon project request, a plan will be made together with the biophysical expert of the facility (Alex Fish) to select the optimal method(s) to address the research question.
The facility hosts a high throughput crystallisation platform for screening and optimisation of protein crystallisation conditions. The facility supports the process up to the level that synchrotron-ready crystals heave been obtained. From that point onwards, the project applicant should consult/collaborate with a crystallographer to prepare for X-ray diffraction data collection at a synchrotron together with crystallography expertise to solve the structure.