Protein Facility

Equipment

The Protein Facility is equipped with all requirements for heterologous expression and purification of recombinant proteins. A variety of biophysical techniques are also available to determine the protein quality and also to perform activity or interaction experiments.

Protein expression systems

  • Bacterial expression. We routinely test a variety of expression strains with all common expression tags. Our expression setup allows for expressions of up to 80 litres.
  • Baculoviral expression. We also have a variety of expression constructs and the means to generate BacMids for protein production using Sf9 insect cells.

Protein purification

We have a range of ‘standard’ affinity beads and columns, compatible with our expression plasmid set. These can use in either a gravity flow set up, as well as using our Bio-rad NGC FPLC systems in a continuous flow.

  • Affinity purification. His-tag (Ni-NTA or TALON), GST-tag or Strep-tag purification is carried out regularly.
  • Ion exchange. Including cation (ResourceS, HiTrap S, PorosS) or anion (ResourceQ, HiTrap and PorosQ) exchange or Heparin columns.
  • Size exclusion. Both small (10/30) and large (16/60, 26/60) preparative scale columns with various bead size (Superdex75, Superdex200/SEC650, Superose6) are available for use on the NGC. Analytical runs: see below.
  • Others. Various other, specialized columns for hydrophobic interactions or chitin-binding are available.

Biophysical analysis

  • Sample purity. Generally this is tested using SDS-PAGE, and can, in some cases, also be addressed using HRMS at the Ovaa lab.
  • TM measurements. Using the Tycho NT 6.1 melting curves for protein quality can be obtained.
  • Analytical chromatography. Most purification columns are available on our ÄKTAµ setup, to allow screening using low protein amounts.
  • MALS. We have a Wyatt setup for Multi Angle Light Scattering, coupled to size-exclusion to obtain protein size and stoichiometry.
  • DLS. We have a Wyatt Nanostar for batch Dynamic Light Scattering measurements.
  • ITC. For isothermal titration calorimetry we have a TA Instruments Affinity available with nanolitre cell volume (190 µL)
  • Spectrophotometric plate reader. For fluorescence based assays we have the BMG LabTech ClarioStar, capable of tuning to any wavelength (230-800 nm).
  • BLI. On our Sartorius Octet96 we are able to perform biolayer interferometry experiments, allowing us to determine on- and off-rates (and thus K¬D) for biomolecular interactions.