VIB Protein Core
Core leader: Ir. Jurgen Haustraete
Tel:+32 9 33 13 610 - Fax:+32 9 33 13 609
The PSF offers solutions for producing mg amounts of research grade proteins, or to optimize expression vectors and production conditions in different systems. When the pure protein is not the endpoint, also Conjugation of the purified protein is possible. The PSF services include scale up of microbial cultures up to 6 or 20 L, mammalian cell culture up to 12 L, and downstream processing and purification adapted to those scales. The deliverable is the end product of your process (usually a protein) and a documentation that each step is carried out to the state of the art. In order to rapidly optimize expression analysis, PSF uses a technology platform for parallel expression analysis with numerous possible combinations, i.e. FastScreen. Expression optimization is offered in Escherichia coli, Pichia pastoris and mammalian cells (HEK293F). Matrix-approach based optimizations are also available for refolding of inclusion bodies, i.e. FastFold, and for purification process optimization, i.e. FastClean. Based on the FastScreen result, a Production can be performed. The FastClean can lead to a Purification strategy to make mg amounts of pure protein. Depending on the purpose of the pure protein; fluorescent labels, polymers, … can be attached by Conjugation. Protein Analytics can be performed to guarantee the quality of the delivered protein. Some Protein Analytics are done in house, others in collaboration with specialized groups or institutes. Not only the purity, concentration or LPS content is important but also the activity or affinity of a protein. Therefor PSF performs also different (cellular) Activity Assays and invests in technologies for Protein-Protein Interactions. Beyond servicing, Research on new technologies and opportunities available on the market is done regularly by the PSF. By the enormous interest in single domain antibodies (also called nanobodies®), PSF has specialized in Nanobody® Production and Purification.
FastScreen is a 3-4 week feasibility study service for Escherichia coli, Pichia pastoris and mammalian cell expression solutions that is routinely used at PSF. At the end, (± 1 month) FastScreen offers a go/no-go decision for expression strategies to our staff. The service presents comparison of several production strategies in a uniform vector frame, allowing true strategy comparison.
Scale up facilities for fermentation of microbial organisms up to 20 liter (fed batch fermentation) and mammalian cells up to 12 liter are available at PSF. Accordingly, standard operating procedures for collection and lysis of cells adapted to these scales are available.
Production in inclusion bodies is a well-established strategy in Escherichia coli. Inclusion bodies are usually more resistant to proteases and thus accumulate to high amounts. Also, after isolation of the inclusion bodies, the protein of interest is already reasonable pure. However, the process of refolding the denatured proteins can be a difficult task, with low yield and laborious methods. PSF offers a 2-3 week study using a matrix screening to find the optimal refolding buffer. This matrix screen is designed in-house. Also some methods of refolding can be compared (dilution, dialysis, on-column).The deliverable is a study and an outline for a refolding process, which can also be validated.
If no purification method is available, a screen can be set up to test a range of different (or combinations of) matrices to purify the protein of interest.
Fluorescent molecules (antibodies) for microscopy are very expensive for VIB research groups. When the hybridoma cell line is available for the production of an antibody, PSF can produce easily mg amounts (1-100 mg) for labeling. A lot of labeling is done with AlexaFluor groups which are easy to use. PSF has also expression vectors available for fusion of proteins to fluorescent proteins (GFP, mCherry). Beside fluorescent labeling; small, very stable molecules like nanobodies®, alphabodies®, affibodies® are becoming more and more important for therapeutic use. A disadvantage of these molecules is their low serum half-life. This can be increased by covalent labeling with all kinds of molecules (like PEG). PSF has also in this field expertise.
After the selection of your nanobodies® by the Nanobody Service Faility (NSF), PSF can help you with the production and purification. PSF has already produced and purified hundreds of nanobodies®: from Escherichia coli, from Pichia pastoris, with fusion tag, without fusion tag, small scale, 20 liter fermenter scale, monovalent, bivalent, fused to cytokines, ... Escherichia coli is still the most used system for the expression of monovalent nanobodies® and the easiest one. The yield varies from nanobody® to nanobody®. When you want to increase the yield, PSF can shift the production from Escherichia coli to Pichia pastoris. In Pichia pastoris is the yield much higher. Also the yield is going down from monovalent to bivalent, trivalent, ... nanobodies® in Escherichia coli. When you start with bivanlent nanobodies®, you have to shift to Pichia pastoris to be good. Also, when you want to express nanobodies® without any affinity tag, Pichia pastoris is the system to use. In this way, you can purify the nanobody® with conventional chromatography ( ion-exchange, hydrophobic interaction, ...). When you start with nanobodies® fused to other proteins like cytokines, mammalian cells (HEK293F) are needed when post-translational modification is important.
Folding of proteins and protein-protein interactions are becoming more and more important. A limited amount of technologies to analyze this are available in PSF and distributed over different VIB research groups (Biacore, SwitchSense, AlphaScreen). PSF wants to invest in the knowledge of these technologies and instruments when their opportunities are proven. Initial projects can be done on demo’s and instruments available in the protein service facility consortium in Europe (P4EU). Important technologies: FACS, CD spectrometry, DLS, SLS, MALS, isothermic titration calometry, microscale thermophoresis, ...
For some proteins, not only the purity, affinity, concentration, … is important but also the activity. Most of these assays are cellular assays. PSF has the knowledge to perform some assays for cytokines (TNFα, IL2, …) . For these cytokine assays, WHO standards are used to refer to.
Available Stock Proteins