Services offered to the researchers at HMGU
At the PEPF we are using and supporting the following cloning technologies:
- Restriction enzymes cloning
- Gateway cloning technology
- Ligase independent cloning (LIC)
- Sequence and ligase independent cloning (SLIC)
- SLiCE cloning (only for in-house projects)
For all these cloning technologies appropriate vectors are available (see Materials).
Small-scale bacterial expression screening
For the small-scale expression screening in Escherichia coli we have developed the so-called Fast Soluble Expression Screen. Small-scale (4 ml) cultures are performed in parallel in a 24-deep well block. Cells are harvested and lyzed by freeze-thaw cycles and expressed proteins are purified over 50-μl columns. The screen is performed to find the best E. coli expression host and optimal expression conditions for a specific target protein.
Large-scale bacterial protein expression
Target proteins are expressed on a large-scale (1-10L) in the best E. coli expression host and under optimal expression conditions.
Insect cell expression
Target proteins are expressed using the baculovirus/insect cell expression system. In small-scale experiments the expression conditions are optimized (cell line, multiplicity-of-infection, culture temperature and duration). The best conditions are used to scale-up protein production. For these experiments we have two cell lines available: Sf21 and Hi5.
At the PEPF we purify over-expressed and native proteins at different scales (from μg to multi mg amounts) using a wide range of chromatographic techniques such as affinity, ion exchange and size exclusion chromatography.
For characterization of targets proteins the following techniques are available:
- Dynamic Light Scattering (DLS) to determine the size and the monodispersity of a target protein.
- Static Light Scattering (SLS) to determine the oligomeric state of a target protein and the stoichiometry of protein-protein and protein-ligand complexes.
- Isothermal Titration Calorimetry to study ligand binding.
- Thermal shift (ThermoFluor) Assay to determine protein stabiliy and study ligand binding and protein function.