The VIB Protein Core offers solutions for producing mg amounts of research grade proteins, or to optimize expression systems and production conditions. When the pure protein is not the endpoint, also conjugation of the purified protein is possible.
The VIB Protein Core 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, the VIB Protein Core 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, CHO). For expression in mammalian cells, the Core can use transient expression (in HEK293F cells) or can provide you with a stable cell line (HEK293F, CHO) expressing your protein of interest. 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 the Core performs also different (cellular) Activity Assays and invests in technologies for Protein-Protein Interactions (Bio-Layer Interferometry, Octet Red). The VIB Protein Core invested also in the SEC/MALS/UV/RI technology to understand even better the folding and monomeric/oligomeric state of your protein in solution. The quality of the recombinant protein is important.
Beyond servicing, Research on new technologies and opportunities available on the market is done regularly by the Core. By the enormous interest in single domain antibodies (also called nanobodies®), the VIB Protein Core has specialized in Nanobody® Production and Purification.
The VIB Protein Core also specialized in the expression of recombinant proteins in mammalian cells. We optimized transient expression conditions in HEK293F cells for first screening expressions and productions. But, expression yield can be increased by making stable cell lines in HEK293F and CHO based on transposon technologies.
The VIB Protein Core is embedded in the VIB-UGent Center for Inflammation Research.
When post-translational modifications in recombinant proteins are important, a mammalian cell line is the system to use for expression. The past years, the Core invested in technology for recombinant expression of proteins in mammalian cells, more especially the expression in FreeStyle 293 cells. We optimized the parameters of standard transient protocols available and adapted it with small tricks, … When this is not enough, transposon based technologies are introduced to make stable cell lines in HEK293F and CHO to reach even higher expression levels.
To find out if the Core performs well in this field, we participated in a European benchmarking study on recombinant expression of proteins in mammalian cells. There were 7 participating laboratories of European academic institutes and companies. The VIB Protein Core came out of this study as one of the two best performing labs, the two labs disassociated themselves from the others. Knowing that the other lab was the protein science group of the Novartis Institute of Biomedical Research, with years of experience in recombinant expression of proteins in mammalians, the VIB Protein Core performed very well.
In 2019, an international evaluation committee postulated: “Eng. Jurgen Haustraete, the group leader of the VIB Protein Core, is an excellent scientist with more than 20 years of experience in the field of protein production and purification. Over the past 15 years he has built up a top level protein production facility. He has created a very stimulating working environment with highly motivated and experienced co-workers. The VIB Protein Core is one of the top academic protein production core facilities in Europe.”
FastScreen is a 3-4 week feasibility study service for Escherichia coli, Pichia pastoris and mammalian cell expression solutions that is routinely used at the VIB Protein Core. 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.
Finally stable cell lines can be made in Pichia pastoris, HEK293F an CHO.
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. The Core 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, the Core can produce easily mg amounts (1-100 mg) for labeling. A lot of labeling is done with AlexaFluor groups which are easy to use. We have 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). The VIB Protein Core also has expertise in this field.
Scale up services for fermentation of microbial organisms up to 20 liter (fed batch fermentation) and mammalian cells up to 12 liter can be done. Accordingly, standard operating procedures for collection and lysis of cells adapted to these scales are available.
After the selection of your nanobodies® by the VIB Nanobody Core, we can help you with the production and purification. The VIB Protein Core 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, we can shift the production from Escherichia coli to Pichia pastoris. In Pichia pastoris, the yield is much higher. Also the yield is decreasing from monovalent to bivalent, trivalent, ... nanobodies® in Escherichia coli. When you start with bivalent nanobodies®, you have to shift to Pichia pastoris to be successful. Furthermore, when expressing nanobodies® without any affinity tag, Pichia pastoris is the system to use. This way, the nanobody® can be purified with conventional chromatography (ion-exchange, hydrophobic interaction, ...). When you start with nanobodies® fused to other proteins like cytokines and Fc’s from IgG’s, mammalian cells (HEK293F, CHO) are needed when post-translational modifications are important.
For many recombinant proteins post-translational modifications are important and even essential. Therefore the VIB Protein Core invested a lot in extracellular, and even intracellular, expression of proteins in mammalian cells. The used systems are HEK293F and CHO because they are respectively human and used a lot in industry. Most people use these cells in a transient way because it is fast. But, the expression levels are now and then disappointing. To overcome this drawback, the Core makes stable cell lines based on transposon technologies, that can increase expression levels with more than a factor 10. Working with inducible promoters can also overcome the fact that some recombinant proteins are toxic for the used expression system.
· Antibody anti-hVEGF (B20)
· Antibody Hamster anti-mCD11C (418)
· Antibody Hamster anti-mCD11C (N418) AlexaFluor700 conjugate
· Antibody Hamster anti-mCD28
· Antibody Hamster anti-mCD28 (37.51)
· Antibody Hamster anti-mCD3e (145-2c11)
· Antibody Mouse anti-hCD3 (OKT3)
· Antibody Rat anti-mCD103 (M290)
· Antibody Rat anti-mCD16/32 (2.4G2)
· Antibody Rat anti-mCD205 (NLDC145)
· Antibody Rat anti-mCD24 (J11D)
· Antibody Rat anti-mCD8α (53-6-7)
· Antibody Rat anti-mIFNγ (AN18)
· Antibody Rat anti-mIL10 (2A5.1)
· Antibody Rat anti-mIL5 (TRFK4)
· Antibody Rat anti-mIL5 (TRFK5)
· Antibody Rat anti-mTNFα (1F3F3D4)
· Antibody anti-SEAP
· Caspase3 (murine)
· FLT3 (human)
· Galectin-10 (human) His N-terminal fusion
· GM-CSF (human)
· GM-CSF (murine)
· HRV-3C protease
· IFNγ (murine)
· IL1β (murine)
· IL1β (murine) His-TEV N-terminal fusion
· IL2 (murine)
· IL22 (murine)
· IL4 (human)
· IL4 (murine) His-Caspase3 N-terminal fusion
· IL6 (human)
· IL7 (human)
· LIF (murine)
· M-CSF (human) Strep C-terminal fusion
· M-CSF (murine)
· R-spondin1 (murine) His C-terminal fusion
· SenP2 protease
· TEV protease
· TNFα (human)
· TNFα (human) FLAG fusion
· TNFα (murine)
· TNFα (murine) FITC conjugate
· VEGF164 (murine)
· VEGF165 (human)
· Ym1 (murine)