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

Murschel, Frédéric; Liberelle, Benoît; St-Laurent, Gilles; Jolicœur, Mario; Durocher, Yves; Crescenzo, Grégory De

doi:10.1016/j.actbio.2013.02.032

Cited by 26 publications

(44 citation statements)

References 49 publications

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“…The creation of such new bio-inspired molecules with enhanced features can find widespread use in a number of biomedical and therapeutic applications (Bromley and Channon 2011). More specifically, the E/K coiled-coil system has proven to be an excellent and versatile tool for (1) the detection of coil-tagged proteins by Western blot (Boucher et al 2010) or ELISA (Liberelle et al 2010a), (2) the coiled-coil-mediated protein dimerization (De Crescenzo et al 2004), as well as (3) the stable and oriented capture of coil-tagged proteins onto various surfaces harboring the partner coil peptide for applications in biosensing (Chao et al 1998;De Crescenzo et al 2003b) and regenerative medicine (Murschel et al 2013;Liberelle et al 2010b;Fortier et al 2013; Abstract A de novo heterodimeric coiled-coil system formed by the association of two synthetic peptides, the Ecoil and Kcoil, has been previously designed and proven to be an excellent and versatile tool for various biotechnology applications.However, based on the challenges encountered during its chemical synthesis, the Kcoil peptide has been designated as a "difficult peptide". In this study, we explore the expression of the Kcoil peptide by a bacterial system as well as its subsequent purification.…”

Section: Introductionmentioning

confidence: 99%

Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif

et al. 2015

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A de novo heterodimeric coiled-coil system formed by the association of two synthetic peptides, the Ecoil and Kcoil, has been previously designed and proven to be an excellent and versatile tool for various biotechnology applications. However, based on the challenges encountered during its chemical synthesis, the Kcoil peptide has been designated as a "difficult peptide". In this study, we explore the expression of the Kcoil peptide by a bacterial system as well as its subsequent purification. The maximum expression level was observed when the peptide was fused to thioredoxin and the optimized purification process consisted of three chromatographic steps: immobilized-metal affinity chromatography followed by cation-exchange chromatography and, finally, a reverse-phase high-performance liquid chromatography. This entire process led to a final volumetric production yield of 1.5 mg of pure Kcoil peptide per liter of bacterial culture, which represents a significant step towards the cost-effective production and application of coiled-coil motifs. Our results thus demonstrate for the first time that bacterial production is a viable alternative to the chemical synthesis of de novo designed coil peptides.

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Section: Introductionmentioning

confidence: 99%

Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif

et al. 2015

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“…[36] Chondroitin sulfate, although known for its anti-apoptotic properties, [48,49] did not induce a significant increase in HUVEC survival when compared to the other controls (Fig. 5).…”

Section: Huvec Survival On E-egf-and E-vegf-decorated Surfaces After mentioning

confidence: 99%

“…On the contrary, the more E-VEGF in the solution, the less captured E-EGF on the surface. It is here noteworthy that E-VEGF was expressed as a homodimeric protein and thus bears 2 Ecoil tags [46], whereas E-EGF is monomeric and has one Ecoil moiety only. [47] Our observations can therefore be explained by an avidity phenomenon that favors the grafting of E-VEGF over the grafting of E-EGF (Fig.…”

Section: Simultaneous Incubation Of E-egf and E-vegf Was Also Testedmentioning

confidence: 99%

“…Of interest, coiled-coil immobilized and free EGF and VEGF led to comparable cell survival when provided at sufficiently high doses. [36,37] Although this system had been limited so far to the immobilization of a single type of GF at a time, we hypothesized that Kcoil-bearing substrates could readily be functionalized with multiple Ecoil-tagged GFs. The objective of this work was therefore to investigate the feasibility of GF co-immobilization, its control and the advantage of the stable coiled-coil capture system as a platform to investigate the collaborative effect between GFs and its potential to create a coating with tailored properties.…”

Section: Introductionmentioning

confidence: 99%

“…[34] Ecoil/Kcoil mediated capture has been successfully applied to tether various Ecoil-tagged GFs, such as the epidermal growth factor (EGF) [35] and the vascular endothelial growth factor (VEGF), [36] onto substrates that had been predecorated with Kcoil peptides. Careful positioning of the Ecoil tags on the chimeras corresponding to each GF allowed this immobilization strategy to preserve the GF bioactivity after tethering (as deduced from receptor activation, [34] increase in adhesion [27], proliferation [27] or survival [36,37] of cells). Of interest, coiled-coil immobilized and free EGF and VEGF led to comparable cell survival when provided at sufficiently high doses.…”

Section: Introductionmentioning

confidence: 99%

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Controlled co-immobilization of EGF and VEGF to optimize vascular cell survival

et al. 2016

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Growth factors (GFs) are potent signaling molecules that act in a coordinated manner in physiological processes such as tissue healing or angiogenesis. Co-immobilizing GFs on materials while preserving their bioactivity still represents a major challenge in the field of tissue regeneration and bioactive implants. In this study, we explore the potential of an oriented immobilization technique based on two high affinity peptides, namely the Ecoil and Kcoil, to allow for the simultaneous capture of the epidermal growth factor (EGF) and the vascular endothelial growth factor (VEGF) on a chondroitin sulfate coating. This glycosaminoglycan layer was selected as it promotes cell adhesion but reduces non-specific adsorption of plasma proteins. We demonstrate here that both Ecoiltagged GFs can be successfully immobilized on chondroitin sulfate surfaces that had been pre-decorated with the Kcoil peptide. As shown by direct ELISA, changing the incubation concentration of the various GFs enabled to control their grafted amount.Moreover, cell survival studies with endothelial and smooth muscle cells confirmed that our oriented tethering strategy preserved GF bioactivity. Of salient interest, coimmobilizing EGF and VEGF led to better cell survival compared to each GF captured alone, suggesting a synergistic effect of these GFs. Altogether, these results demonstrate the potential of coiled-coil oriented GF tethering for the co-immobilization of macromolecules; it thus open the way to the generation of biomaterials surfaces with fine-tuned biological properties.

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A low‐temperature SPR‐based assay for monoclonal antibody galactosylation and fucosylation assessment using FcγRIIA/B

Gaudreault,

Forest‐Nault,

Gilbert

et al. 2024

Biotech & Bioengineering

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Monoclonal antibodies (MAbs) are powerful therapeutic tools in modern medicine and represent a rapidly expanding multibillion USD market. While bioprocesses are generally well understood and optimized for MAbs, online quality control remains challenging. Notably, N‐glycosylation is a critical quality attribute of MAbs as it affects binding to Fcγ receptors (FcγRs), impacting the efficacy and safety of MAbs. Traditional N‐glycosylation characterization methods are ill‐suited for online monitoring of a bioreactor; in contrast, surface plasmon resonance (SPR) represents a promising avenue, as SPR biosensors can record MAb–FcγR interactions in real‐time and without labeling. In this study, we produced five lots of differentially glycosylated Trastuzumab (TZM) and finely characterized their glycosylation profile by HILIC‐UPLC chromatography. We then compared the interaction kinetics of these MAb lots with four FcγRs including FcγRIIA and FcγRIIB at 5°C and 25°C. When interacting with FcγRIIA/B at low temperature, the differentially glycosylated MAb lots exhibited distinct kinetic behaviors, contrary to room‐temperature experiments. Galactosylated TZM (1) and core fucosylated TZM (2) could be discriminated and even quantified using an analytical technique based on the area under the curve of the signal recorded during the dissociation phase of a SPR sensorgram describing the interaction with FcγRIIA (1) or FcγRII2B (2). Because of the rapidity of the proposed method (<5 min per measurement) and the small sample concentration it requires (as low as 30 nM, exact concentration not required), it could be a valuable process analytical technology for MAb glycosylation monitoring.

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

Cited by 26 publications

References 49 publications

Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif

Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif

Controlled co-immobilization of EGF and VEGF to optimize vascular cell survival

A low‐temperature SPR‐based assay for monoclonal antibody galactosylation and fucosylation assessment using FcγRIIA/B

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