The NKI Protein facility is a protein production and characterization facility at the Division of Biochemistry, The Netherlands Cancer Institute (NKI)
The Facility has access to a high-throughput macromolecular crystallisation platform. Liquid handling robots are available for preparation of nanoliter crystallisation drops in 96-well format, including a 96-well pipetting robot (CyBi-SELMA) for filling up reservoir wells and a Mosquito for creating nanoliter crystallisation drops. Crystallisation trials can be stored and imaged by two RockImager systems, one at 4 °C and one at 20 °C. When suitable crystals are obtained for x-ray diffraction analysis, the applicant has to collaborate with a crystallography facility. Solving crystal structures is beyond the scope of the facility.
Depending on the project, the platform for expression will be chosen. Proteins can be expressed in E. coli, Insect cells and mammalian cells. For each of the systems we can do small scale (few ml) as well as large scale (few liters) production. For insect cell expression SF9 and SF21 cells are used for expression of intracellular and extracellular proteins. The cells are grown in suspension cultures in volumes up to 500 ml/flask. We use Baculovirus technology for over-expression of proteins in insect cells.
Depending on the project, the platform for expression will be chosen. Proteins can be expressed in E. coli, Insect cells and mammalian cells. For each of the systems we can do small scale (few ml) as well as large scale (few liters) production. Our mammalian expression system has currently been optimized for HEK-293T cells and is typically used for expression of extracellular (secreted) proteins. These cells can be cultured in flasks, dishes or rollerbottles. A glycosylation deficient strain - gnT1-deficient HEK293S - is also available for production of eukaryotic proteins that would benefit from removal of glycosyl chains (e.g. for crystallisation purposes). The use of other cells for expression can be discussed.
The NKI Protein Facility (external website) is part of the Instruct-NL national Instruct Centre in the Netherlands and provides support for the production, purification, biophysical characterisation and crystallisation of proteins. It is accessible to both internal (NKI)- and external research groups. In particular researchers who don't have the experience or the facilities to produce and analyze proteins can request for assistance with the design and performance of the experiments. Some experiments (e.g. high throughput macromolecular crystallisation) will be performed by the facility members and are offered as a service. A particular strength of the facility is in the characterisation of macromolecular interaction with a multitude of biophysical methods. Important: to ensure a smooth application process, we strongly recommend any users to contact us before submitting an application.
Expression of soluble,functional proteins is dependent on many different factors. The facility can assist in the design of selective protein fragments using structural- and bio-informatics tools to increase the chance for success.
The facility has platforms for protein expression in E. coli (large-scale incubators at different temperatures), in insect cells using the baculovirus system, and for protein expression in mammalian cells (large roller-bottle incubator with a capacity for 90 bottles).
A variety of chromatography steps - including affinity, ion exchange and size exclusion chromatography - can be performed on different Akta and BioRad systems.
The facility offers a variety of biophysical techniques to characterise macromolecular properties and interactions. These include:
The facility has access to an automated high-throughput crystallization screening platform. Crystallization conditions can be screened for in 96-well format in 100-200 nanoliter droplets. Plates are prepared by (nanoliter) liquid handling robots and crystallization trials are visualized over time by automated imaging systems at 4 °C and 20 °C; results are accessible via web-servers.
The Netherlands Cancer Institute/Antoni van Leeuwenhoek hospital (NKI/AvL) is conveniently situated at the south-west side of Amsterdam. From Schiphol airport It is about 15 minutes drive to the institute (bus or car) or 7 minutes by train (to Amsterdam Lelylaan station, followed by a 15 min. walk). From the institute it is easy to connect to Amsterdam city centre via tram, bus or train. More details can be found at the NKi website:
Visit this centre and more by submitting a proposal for access.Submit Proposal