The research department of the European Institute of Oncology (IEO) is a multidisciplinary center for cancer research, which hosts about 20 groups interested in basic and translational aspects of epigenetics, genomics, cell and stem cell biology. Despite being a very young Institute, IEO is internationally recognized as a major research center and receive funding from several agencies including the Italian Ministry of Health, the Italian Association for Cancer Research and the European Commission. The scientific groups of the department have access to a number of centralized core facilities allowing cell culture, next-generation sequencing, imaging and crystallography with forefront instrumentation.

Within Instruct-ITALIA, the IEO facility  is represented by the Crystallography Unit in synergy with the research groups focusing on Structural Biology.

Platforms available for access through Instruct-ITALIA:

• Protein production in insect and mammalian cells
• High throughput crystallization
• Protein Characterisation and Affinity Determination Assays
• Sample preparation for cryo-EM analyses

Techniques

Protein production in insect and mammalian cells

The baculovirus expression system permits high levels of expression of recombinant proteins in insect cells. With respect to bacteria, it has the advantages of allowing production of larger targets, which will get properly post-translationally modified by the eukaryotic cell. We express proteins and multi-protein complexes in insect cells with the MultiBac and biGBac systems, and offer a complete service that goes from gene cloning to protein production. For challenging targets requiring human-specific PTMs, transient transfection of HEK293 cells grown in suspension will be used in combination with a dedicated plasmid library that allows the rapid screening of different tags type.

High throughput crystallization

We manage an automated platform for the high-throughput crystallization of macromolecular samples, in order to maximize the chances of crystallization. Initial trials are set up using a nano-dispenser robot, allowing screening of 384 conditions with as little as 50/60 μl of sample. Crystallisation plates are automatically imaged on a standard schedule, and drop images are available online, allowing remote crystal appearance and growth monitoring. Optimization of initial hits is tipically performed manually in 24 well plates with the hanging drop method in 2 μl drops. In this cases, tailored screens are prepared based on initial hits conditions. The Unit offers a complete service, from trials set up to crystal optimization, or simple access to reagents and instrumentation for expert users.

Protein Characterisation and Affinity Determination Assays

The Unit provides the experimental determination of the oligomeric state of proteins in solution via Static Light Scattering and the measurement of binding affinities of macromolecular complexes via Isothermal Titration Calorimetry, BLI and fluorescence polarization.

Sample preparation for cryo-EM analyses

We are equipped with a Vitrobot Mark IV for cryoEM grid plunging prior to screening. A pool of grids can be selected depending on the sample and users’ requirements. CryoEM data are processed in-house using the most updated software for cryoEM data processing installed on a dedicated multi-core and multi-GPU workstation.