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Welcome to the homepage of the Protein Expression and Purification Facility

The protein Expression and Purification Facility (PEPF) was established to help you with all aspects of the production of recombinant proteins.

Cloning
Production of expression constructs is usually done by the users. We can help you to decide which construct(s) to make, which vector(s) to use and how to clone your gene(s) of interest into these vectors. We mainly use restriction enzyme and assembly cloning methods but also support the Gateway cloning technology and ligase independent cloning (LIC) methods. Specific cloning and expression vectors are available for all of the above mentioned cloning technologies (see Materials).
All expression constructs have to be sequenced before handing them to us.

Expression screening
To find optimal expression conditions (such as construct, medium, temperature) we perform small-scale screening. The cultures (4 ml) are done in parallel in a 24-deep well block. After harvesting the cells are lyzed by freeze-thaw cycles and the expressed, soluble proteins purified over a small column (usually 50 μl) of the appropriate resin. In most cases an IMAC (immobilized metal affinity chromatography) resin (for instance Ni-NTA) is used but also other affinity chromatography (such Glutathione-Sepharose) or ion exchange (such a S- or Q-Sepharose) resins can be used. In the case of IMAC purification the constructs are required to have an N-or C-terminal His-tag.

Protein expression in Escherichia coli
Target proteins are expressed under the optimal conditions determined by small-scale expression screening. The expression cultures are normally performed in 2-L flasks in a shaker-incubator (0.5-10L) using ZYM 5052 auto-induction medium (Studier, 2005). Other media such a lysogeny broth (LB) and terrific broth (TB) are also used.

Protein expression in insect cells
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 (0.1-2L). At the moment two cell lines are used: Sf21 and Hi5.

Protein purification
After harvesting and cell lysis the expressed proteins can be purified using a wide range of chromatographic techniques such as affinity, ion exchange and size exclusion chromatography. For every individual protein the purification protocol has to be optimized. Since most proteins contain an N-terminal His-tag the defaults starting protocol is as follows:

Capture of the target protein by IMAC.
Removal of the His-tag (and a potential protein tag) by protease cleavage (using TEV or 3C protease).
Removal of the tag(s), uncleaved target protein and impurities by a second IMAC step.
Polishing of the final preparation and removal of protein aggregates by size exclusion chromatography (SEC).

In most cases one of more of these steps need to optimized and other steps need to be added in order to produce the high quality proteins needed for the different applications.
Most purification steps are performed using an Äkta Purifier system (GE Healthcare) of which there are several in the lab. For this system a wide range of purification columns are available.

Protein quality assessment
High quality protein is needed for nearly all the possible applications. Many experiments have failed or proved to be irreproducible because poor protein preparation were used. Therefore, it is important to assess the quality of the produced recombinant proteins. A minimal set of parameters that should be tested has been suggested (by the ARBRE-MOBIEU and P4EU networks).

Purity - usually tested by SDS-PAGE
Homogeneity (aggregation state) - preferably checked by size exclusion chromatography (SEC) or dynamic light scattering (DLS).
Identity - checked by intact protein mass or peptide mass fingerprinting.

UV-spectrum - especially for proteins that bind nucleotides.
Conformational state
- determined by circular dichroism (CD), different scanning calorimetry (DSF) or NMR.

Protein characterization
Different techniques are available for the characterization of the produced proteins.

Dynamic Light Scattering (DLS)
Determination of the size and espcially the monodispersity of a protein.
Static Light Scattering (SLS)
Determination of the accurate mass of a protein or protein complex. From the value it is possible to calculate the oligomeric state of a protein and the stoichiometry of protein-protein and protein-ligand complexes.
Isothermal Titration Calorimarty (ITC)
Determination of the stoichiometry and affinity of protein-protein and protein-ligand interactions.
Thermal shift (thermoFluor) assays
Determination of the thermal stability of a protein. This method can be used to screen many conditions in order to find the optimal buffer for a specific protein. Furthermore, it can be used to study protein-ligand interactions.

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