Gilles St-Laurent

The Bioactivity and Receptor Affinity of Recombinant Tagged EGF Designed for Tissue Engineering Applications Is Defined by the Nature and Position of the Tags

For tissue engineering applications, growth factor immobilization on cell culture scaffolds bears the potential to stimulate cell proliferation while minimizing costs associated to soluble growth factor supply. In order to evaluate the potential of a de novo-designed heterodimerization peptide pair, namely the E and K coils, for epidermal growth factor (EGF) grafting on various scaffolds, production of coil-tagged EGF chimeras using a mammalian cell expression system as well as their purification have been performed. The influence of the type of coil (E or K) upon EGF bioactivity, assessed in an in vitro cell assay, was compared to that of the fragment crystallizable (Fc) domain of immunoglobulin G by monitoring phosphorylation of EGF receptor (EGFR) upon chimeric EGF exposure. Our results demonstrate that the type and the location of the tag have a strong impact on growth factor bioactivity (EC50 ranging from 5.5 to 63 nM). Additional surface plasmon resonance-based biosensor experiments were conducted to test the ability of captured chimeric EGF to bind to their receptor ectodomain in vitro. These experiments indicated that the oriented coiled-coil-mediated immobilization of EGF was significantly more efficient than a random approach as coil-tagged EGF displayed enhanced affinities for artificially dimerized EGFR ectodomain when compared to Fc-tagged EGF (apparent KD of 5 pM vs. 16 nM). Altogether, our results highly suggest that coil-tagged chimeras represent an attractive avenue for the oriented immobilization of growth factors for tissue engineering applications and that HEK293 cells offer a robust platform for their expression in a bioactive form.

Stable expression of chimeric heavy chain antibodies in CHO cells.

Camelid single domain antibodies fused to noncamelid Fc regions, also called chimeric heavy chain antibodies (cHCAb), offer great potential as therapeutic and diagnostic candidates due to their relatively small size (80 kDa) and intact Fc. In this chapter, we describe two approaches, limiting dilution and minipools, for generating nonamplified Chinese hamster ovary cell lines stably expressing cHCAb in suspension and serum-free cultures using a stringent antibiotic selection. Neither of the protocols necessitates the acquisition or implementation of expensive automated infrastructures and thus could be applied in any lab with minimal cell culture setup. The given protocol allows the isolation of stable clones capable of generating up to 100 mg/L of antibody in batch mode performed in shaker flasks.

Protein detection by Western blot via coiled–coil interactions

We propose an approach for the detection of proteins by Western blot that takes advantage of the high-affinity interaction occurring between two de novo designed peptides, the E and K coils. As a model system, K coil-tagged epidermal growth factor (EGF) was revealed with secreted alkaline phosphatase (SeAP) tagged with E coil (SeAP-Ecoil) as well as with biotinylated E coil. In that respect, we first produced purified SeAP-Ecoil and verified its ability to interact with K coil peptides by surface plasmon resonance biosensing. We demonstrated that protein detection with Ecoil-biotin was more specific than with SeAP-Ecoil. We then showed that our approach is as sensitive as conventional detection strategies relying on nickel-nitrilotriacetic acid-horseradish peroxidase (Ni-NTA-HRP), anti-His-HRP, or anti-EGF. Altogether, our results indicate that the E/K coiled-coil system is a good alternative for protein detection by Western blot.

Coiled-coil-mediated grafting of bioactive vascular endothelial growth factor

Chimeric growth factors may represent a powerful alternative to their natural counterparts for the functionalization of tissue-engineered scaffolds and applications in regenerative medicine. Their rational design should provide a simple, readily scalable production strategy while improving retention at the site of action. In that endeavor, we here report the synthesis of a chimeric protein corresponding to human vascular endothelial growth factor 165 being N-terminally fused to an E5 peptide tag (E5-VEGF). E5-VEGF was successfully expressed as a homodimer in mammalian cells. Following affinity purification, in vitro surface plasmon resonance biosensing and cell survival assays confirmed diffusible E5-VEGF ability to bind to its receptor ectodomains, while observed morphological phenotypes confirmed its anti-apoptotic features. Additional surface plasmon resonance assays highlighted that E5-VEGF could be specifically captured with high stability when interacting with covalently immobilized K5 peptide (a synthetic peptide designed to bind to the E5 moiety of chimeric hVEGF). This immobilization strategy was applied to glass substrates and chimeric hVEGF was shown to be maintained in a functionally active state following capture. Altogether, our data demonstrated that stable hVEGF capture can be performed via coiled-coil interactions without impacting hVEGF bioactivity, thus opening up the way to future applications in the field of tissue engineering and regenerative medicine.

Transient Gene Expression in Serum-Free Suspension-Growing Mammalian Cells for the Production of Foot-and-Mouth Disease Virus Empty Capsids

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.

Biotinylation of the Fcγ receptor ectodomains by mammalian cell co-transfection: application to the development of a surface plasmon resonance-based assay.

We here report the production of four biotinylated Fcγ receptor (FcγR) ectodomains and their subsequent stable capture on streptavidin‐biosensor surfaces. For receptor biotinylation, we first describe an in‐cell protocol based on the co‐transfection of two plasmids corresponding to one of the FcγR ectodomains and the BirA enzyme in mammalian cells. This strategy is compared with a standard sequential in vitro enzymatic biotinylation with respect to biotinylation level and yield. Biotinylated FcγR ectodomains that have been prepared with both strategies are then compared by analytical ultracentrifugation and surface plasmon resonance (SPR) analyses. Overall, we demonstrate that in‐cell biotinylation is an interesting alternative to standard biotinylation protocol, as it requires less purification steps while yielding higher titers.