The Biochemistry and Structural Biology Unit offers the following services
Common Reagents for the Department
The Unit ensure production or assistance in purification of selected enzymes of common usage for the Department, such as Tn5 transposase, TAT-Cre recombinase, 3C protease, Cas9, etc...
Protein Expression and Purification Strategies
The Unit provides expert advice in construct design, protein expression and purification; manages protein production in insect cells (baculovirus expression system), production of secreted proteins in insect cells and HEK cells.
The Unit provides assistance in analysis of secondary and tertiary structure predictions, as well as in strategies of protein expression and purification. Experience in proteins co-expression and complex purifications might be of paramount importance in studying molecular mechanisms of complex biological systems.
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 Bac-to-Bac and GoldenBac system, and offer a complete service that goes from gene cloning to protein production.
Protein Characterisation and Affinity Determination Assays
The Unit provides service and instrumentation for the measurement of binding affinities of macromolecular complexes (ITC and Fluorescence Polarization assays), the determination of oligomeric state of proteins in solution (Static Light Scattering) and the identification of optimal buffer/ligand conditions (Thermal Shift assays).
Crystallogenesis and X-ray Structure Determination
We manage an automated platform for the high-throughput crystallization of macromolecular samples.
Initial crystallization trials are set up using a nano-dispenser robot, allowing screening of 384 conditions with as little as ca. 60 μl of sample. Crystallisation plates are automatically imaged on a standard schedule, and drop images are available online to users, who can follow remotely crystal appearance and growth. Optimization of initial hits is typically 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.
Access to synchrotron light sources for crystal X-ray diffraction data collection is ensured by the Unit thanks to the peer-reviewed allocation of beamtime at the European Synchrotron Radiation Facility (ESRF -Grenoble -F) and Swiss Light Source (SLS - Villigen - CH) beamlines, routinely obtained by the Unit, in collaboration with the scientific group of Marina Mapelli. Data are processed in-house with crystallographic software suites installed and maintained by the Unit with the help of the IEO ICT service.