Protein expression and purification projects
Available host organisms for protein expression in PEPCF:
- Escherichia coli
- Baculovirus-mediated expression in insect cells (Sf9, Sf21 and Hi5 cells)
Currently we do not offer protein expression in other host organisms (such as e.g. yeast and mammalian cells), but we can purify proteins expressed in other host organisms if you are able to provide us with the pellet of the expression cells or with the cell culture supernatant in case of secreted proteins.
We kindly request that you contact us first by phone or e-mail to discuss new expression and purification projects. In case expression constructs and/or protocols are already present, you can immediately fill out our protein expression and purification request form and send the completed form back to us. Please note that this is not an order form and that we can only confirm the acceptance of new projects after a personal discussion. Regarding timing we generally work on a first come, first served basis. Although we prioritize internal EMBL users, we also accept external projects as long as capacity allows it.
In case no pre-existing expression constructs and/or protocols are available, we can design a strategy together with you. To do this as efficiently as possible, it is important for us to have some basic information about the protein:
- Is it a single- or multi-domain protein? Do you want to express the full length protein or separate domains?
- What is the cellular localization of the protein? Does the native sequence contain e.g. a signal sequence or transmembrane domains?
- Does the protein require co-factors for stability or functionality?
- Do you know whether there are disulfide bonds present in the native protein?
- Does the protein require interaction partners for stability (=> co-expression)?
- Does the native protein have any post-translational modifications? If yes, are the native post-translational modifications required for your downstream experiments?
- What are the downstream experiments you want to use the purified protein for?
We do not offer any cloning services, but we do maintain a large collection of expression vectors. All non-commercial expression vectors are also available to external academic users via a Material Transfer Agreement. For expression in E. coli, we accept all commonly used expression vectors. For baculovirus-mediated expression in insect cells, we require expression vectors compatible with the Bac-to-Bac system (e.g. the commercially available pFastBac or MultiBac vectors).
E. coli protein expression and purification
After we receive the sequence-verified expression constructs, we will first perform small scale expression and purification tests in which we assess the expression level, the solubility and the protein enrichment after a small scale affinity purification. Once suitable conditions have been identified, we proceed to large scale expression (1-6 liter per construct) and protein purification using a variety of chromatographic techniques.
Insect cell protein expression and purification
After we receive the sequence-verified expression constructs, we will first generate the recombinant baculoviruses. For transposition of the gene of interest into the baculoviral DNA, we use E. coli DH10EMBacY cells. After isolation of the baculoviral DNA, we perform a bacmid PCR to verify the insertion of the gene of interest into the baculoviral DNA. Sf21 cells are used for the generation of the recombinant baculoviruses. After harvesting the V1 virus, we perform small scale expression and purification tests, in which we assess the expression level, the solubility and the protein enrichment after a small scale affinity purification. For protein expression we use either the Sf21 either the Hi5 insect cell lines. Once suitable conditions have been identified in the small scale tests, we can proceed to large scale expression (1-5 liter per construct) and protein purification using a variety of chromatographic techniques.
Usage of the insect cell culture room (room 351) - internal users only
New users that want to access the insect cell culture room (room 351) are kindly asked to contact the PEPCF staff. After you have received an introduction from a PEPCF staff member, we will give you access to the room via the card reader and add you to the user list. Usage of the room can then be booked online via the PPMS system.
Aliquots of frequently used proteins - internal users only
Aliquots of our frequently used proteins (proteases, polymerases, LIF, Cas9, Tn5, …) can be picked up at any time in the facility (room 303). Please bring some (dry) ice to transport your aliquots. The users from the out-stations can contact us via phone or e-mail and we will then ship the required proteins to the designated groups. A complete overview of all frequently used proteins we provide can be found in the price list.
The PEPCF scientific officer for biophysical characterization (Kathryn Perez) offers assistance with the design, execution and data analysis of various types of biophysical experiments. New users can contact Kathryn Perez directly for a discussion of their experiments and to receive more information regarding the amounts of sample and the buffer conditions that are required for the different biophysical techniques. Please also fill out the biophysical characterization request form and send the completed form back to us. If capacity allows it, we also provide assistance with biophysical characterization for external users. However, external users are kindly requested to contact us first regarding the feasibility of their project and to receive a cost estimate.
Currently available biophysical instruments:
- Isothermal Titration Calorimetry (Mavern PEAQ ITC)
- Circular Dichroism spectrometer (Jasco J815)
- Fluorescence spectrometer (Jasco FP8500)
- UV-VIS spectrophotometer (Agilent Cary60)
- Bio-Tek Synergy 4 microplate reader
- Dynamic Light Scattering (Malvern Zetasizer µV)
- Stopped flow (BioLogic)
- Monolith NT.115 for microscale thermophoresis measurements (NanoTemper)
- Prometheus NT.48 with backscattering optics for Nano-DSF measurements (NanoTemper)
- CFX Connect Real Time PCR system for thermofluor assays (BioRad)