References of "Vandevenne, Marylène"
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See detailProtein-polysaccharide complexes, a tool for protein delivery in CaCO3 microparticles
Ramalapa, Bathabile ULiege; Crasson, Oscar ULiege; Vandevenne, Marylène ULiege et al

Poster (2017, September 06)

INTRODUCTION The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated. The success of these carriers has been driven by the ionic interactions ... [more ▼]

INTRODUCTION The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated. The success of these carriers has been driven by the ionic interactions between proteins and particles making the encapsulation of proteins highly dependent on the pH of reaction solutions and the isoelectric point of the protein.1 This poses a great limitation on the successful loading of proteins into microparticles. In this study, we explored the use of polysaccharide-protein interactions to strongly enhance the encapsulation of proteins in CaCO3 microparticles. EXPERIMENTAL METHODS Previously, Vandevenne and colleagues2 inserted a human chitin binding domain (ChBD) that has intrinsic affinity for hyaluronic acid (HA) into β-lactamase (BlaP). This generated chimeric protein, named BlaPChBD, was shown to be fully bifunctional. In this study this hybrid protein (BlapChBD) was associated to HA and successfully loaded into CaCO3 microparticles using super critical CO2 technology aided by the templating effect of HA on CaCO3. Furthermore, thrombin cleavage sites were engineered on both sides of the inserted ChBD in the chimeric BlaP so that release of the protein from the microparticles could be easily achieved by protease cleavage. The microparticles were characterised for size, surface charge, poly-morphism and protein loading and in-vitro release assays were performed. RESULTS AND DISCUSSION The presence of ChBD inserted into the β-lactamase increased the encapsulation of the protein by 6-fold when complexed with HA (Fig. 1). In addition, we also showed that the encapsulated BlaP remains stable during this process using kinetic reaction of β-lactam hydrolysis. Our data showed that vaterite CaCO3 microparticles of sizes ranging between 4 and 7 µm were produced. We were also able to demonstrate that thrombin cleavage increased the release of the protein from the microparticles within 36 hours from <25% to 87% (Fig. 2). In conclusion, the presence of ChBD successfully improved the encapsulation yield of the protein while retaining up to 81% of its activity. CONCLUSION Protein-polysaccharide complexation demonstrates an excellent approach for the delivery of sensitive biomacromolecules which can otherwise be complicated due to electrostatic hindrances. Future prospects include using the methods we have developed for encapsulation of therapeutic proteins and using calcium carbonate as a carrier and scaffold in bone regeneration for example. [less ▲]

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See detailProtein–polysaccharide complexes for enhanced protein delivery in hyaluronic acid templated calcium carbonate microparticles
Ramalapa, Bathabile ULiege; Crasson, Oscar ULiege; Vandevenne, Marylène ULiege et al

in Journal of Materials Chemistry B (2017), 5

The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated. The success of these carriers is driven by ionic interactions between the encapsulated ... [more ▼]

The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated. The success of these carriers is driven by ionic interactions between the encapsulated proteins and the particles. This poses a great limitation on the successful loading of proteins that have no ionic affinity to CaCO3. In this study, we explored the use of polysaccharide–protein interactions to strongly enhance the encapsulation of proteins in CaCO3 microparticles. Previously, Vandevenne and colleagues inserted a human chitin binding domain (ChBD) that has intrinsic affinity for hyaluronic acid (HA) into a β-lactamase (BlaP). This generated chimeric protein, named BlaPChBD, was shown to be fully bifunctional. In this study we showed that this hybrid protein can associate with HA and be successfully loaded into vaterite CaCO3 microparticles using supercritical CO2 (ScCO2) technology aided by the templating effect of HA on CaCO3. The presence of ChBD inserted into BlaP increased the encapsulation of the protein by 6-fold when complexed with HA. Furthermore, thrombin cleavage sites were engineered on both sides of the inserted ChBD in the chimeric BlaP to achieve release of the protein from the microparticles by protease cleavage. Our results showed that thrombin cleavage increased the release of the protein from the microparticles within 36 hours from <20% to 87%. In conclusion, the presence of ChBD successfully improved the encapsulation yield of the protein while retaining up to 82% of its activity and efficient release of the protein from the microparticles was achieved by protease cleavage. [less ▲]

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See detailProtein-polysaccharide complexes for improved protein delivery within CaCO3 microparticles
Ramalapa, Bathabile ULiege; Crasson, Oscar ULiege; Vandevenne, Marylène ULiege et al

Conference (2017, July 17)

Introduction: The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated due to accessibility, low cost, safety, pH-sensitive properties, high surface ... [more ▼]

Introduction: The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated due to accessibility, low cost, safety, pH-sensitive properties, high surface area and high porosity. The success of these carriers has been driven by the ionic interactions between proteins and particles making the encapsulation of proteins highly dependent on the pH of reaction solutions and the isoelectric point of the protein. This poses a great limitation on the successful loading of proteins into microparticles. In this study we explored the use of polysaccharide-protein complexes to enhance the encapsulation of otherwise poorly encapsulated proteins in CaCO3 microparticles. Methods: A chitin binding domain (ChBD) was inserted on the protein β-lactamase to form a chimeric protein. A protein-polysaccharide complex was formed between the protein and hyaluronic acid (HA) owing to the intrinsic affinity of the ChBD to HA. The chimeric protein was then loaded into CaCO3 microparticles using super critical CO2 technology aided by the templating effect of HA on CaCO3. The microparticles were characterised for size, surface charge, polymorphism and protein loading. Bioactivity and stability of the encapsulated β-lactamase was characterised by kinetic reaction with nitrocefin. A thrombin cleavage site was inserted onto the gene sequence of the protein to achieve release of the protein from the microparticles by proteases mediation using thrombin. Results: Vaterite CaCO3 microparticles of sizes ranging between 6 and 8 µm were produced. The presence of the ChBD on the β-lactamase increased the encapsulation of the protein by 6 fold when complexed with HA. Thrombin mediated release increased the release of the protein from the microparticles within 36 hours from <25% to 87%. The protein-polysaccharide complex demonstrated success in encapsulation of the protein while retaining up to 81% activity of the protein and allowing controlled release by proteases. Conclusion: Protein-polysaccharide complexation demonstrates an excellent approach for the delivery of sensitive biomacromolecules which can otherwise be complicated due to electrostatic hindrances. Future prospects include using the methods we have developed for encapsulation of therapeutic proteins and using calcium carbonate as a carrier and scaffold for example in bone regeneration. [less ▲]

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See detailImproved encapsulation of proteins within calcium carbonate microparticles by means of protein-polysaccharide complexes
Ramalapa, Bathabile ULiege; Crasson, Oscar ULiege; Vandevenne, Marylène ULiege et al

Poster (2017, May 30)

The controlled delivery of proteins within calcium carbonate particles is currently widely investigated. The success of these carriers has been driven by the ionic interactions between proteins and ... [more ▼]

The controlled delivery of proteins within calcium carbonate particles is currently widely investigated. The success of these carriers has been driven by the ionic interactions between proteins and particles making the encapsulation of proteins highly dependent on the pH of reaction solutions and the isoelectric point of the protein. This poses a great limitation on the successful loading of proteins into microparticles. In this study we explored the use of polysaccharide-protein complexes to enhance the encapsulation of otherwise poorly encapsulated proteins in CaCO3 microparticles. A chitin binding domain (ChBD) was inserted on the protein β-lactamase to form a chimeric protein. A protein-polysaccharide complex was formed between the protein and hyaluronic acid (HA) owing to the intrinsic affinity of the ChBD to HA. The chimeric protein was then loaded into calcium carbonate (CaCO3) microparticles using super critical CO2 technology aided by the templating effect of HA on CaCO3. The microparticles were characterised for size, surface charge, poly-morphism and protein loading. Biochemical stability of the encapsulated β-lactamase was characterized by kinetic reaction with nitrocefin. The presence of the ChBD on the β-lactamase increased the encapsulation of the protein by 6 fold when complexed with HA. Thrombin mediated release increased the release of the protein from the microparticles within 36 hours from <25% to 87%. The protein-polysaccharide complex proved successful in enhancing the encapsulation of protein while retaining up to 81% activity and allowing controlled release of the protein by proteases. [less ▲]

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See detailRNA-binding protein engineering for the benefits of research and therapy
Vandevenne, Marylène ULiege

Scientific conference (2017, March 08)

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See detailExploring potentialities of RanbP2-type Zinc Fingers in RNA-binding proteins design
De Franco, Simona ULiege; Vandenameele, Julie ULiege; Galleni, Moreno ULiege et al

Poster (2016, June 29)

Transcriptomes consist of several classes of RNAs, whose roles are central to innumerable biological processes as well as diseases. These observations justify the increasing interest in the engineering of ... [more ▼]

Transcriptomes consist of several classes of RNAs, whose roles are central to innumerable biological processes as well as diseases. These observations justify the increasing interest in the engineering of functionalized RNA-binding proteins (RBPs) to specifically manipulate RNA function. In this context, the RanBP2-type Zinc Finger (ZF) domain emerged as a suitable scaffold for single-stranded RBPs design. The present study aimed to identify the sequence-specificity of several naturally occurring RanBP2-type ZFs by in vitro selection and use the natural variation in their substrate to create arrays of ZF domains displaying customized specificity. A parallel kinetic and thermodynamic characterization of ssRNA-ZFs interaction was performed by Isothermal Titration Calorimetry (ITC) and BioLayer Interferometry (BLI), respectively. Although our data showed that only little variations in the recognized sequence could be observed for the studied ZFs, they contribute to elucidate the molecular basis for the RanBP2-type ZF-ssRNA interaction. [less ▲]

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See detailThe use of protein-polysaccharide complexes to enhance protein delivery in CaCO3 microparticles
Ramalapa, Bathabile ULiege; Crasson, Oscar ULiege; Vandevenne, Marylène ULiege et al

Conference (2016, March 22)

The role of protein therapeutics in modern medicine has increased considerably due to their potential to perform complex functions in the body that cannot be mimicked by simple chemical compounds. To ... [more ▼]

The role of protein therapeutics in modern medicine has increased considerably due to their potential to perform complex functions in the body that cannot be mimicked by simple chemical compounds. To increase the safety, stability and efficacy of protein therapeutics; there exists a need for delivery systems that are biocompatible, prevent premature degradation of bioactives and allow targeted delivery and controlled release. Calcium carbonate (CaCO3) has gained great favour for employment in protein delivery due to possibilities of controlling size, morphology and crystalline forms of particles by tuning the synthesis conditions. The aim of the present work was to assess the significance of polysaccharide-protein complexes in enhancing the encapsulation of proteins in CaCO3 microspheres. A Chitin Binding Domain (ChBD), reported to have affinity for hyaluronic acid, was inserted on β-lactamase enzyme to develop a chimeric protein. The chimeric protein retained the activity of the enzyme and the binding properties and was encapsulated in CaCO3 microspheres by a super critical CO2 (ScCO2) process using hyaluronic acid as a templating agent. The particles were characterised in terms of size, zeta potential, morphology and protein loading. The results obtained confirmed the affinity of the ChBD to hyaluronic acid towards the production of stable, vaterite microparticles. Protein assays demonstrated that the ChBD enhanced the encapsulation of protein by up to 10 fold. Confocal images also suggested high encapsulation of the chimeric protein compared to native protein. Thus the production of polysaccharide-protein complexes seems effective in enhancing the encapsulation of proteins in CaCO3 microparticles using the ScCO2 process. [less ▲]

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See detailThe yeast transcription elongation factor Spt4/5 is a sequence-specific RNA binding protein
Blythe, Amanda; Yazar-Klosinski, Bera; Webster, Michael et al

in Protein Science : A Publication of the Protein Society (2016), 25(9), 1710-1721

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See detailThe unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools.
Figueroa Yévenes, Maximiliano ULiege; Sleutel, Mike; Vandevenne, Marylène ULiege et al

in Journal of structural biology (2016)

Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the ... [more ▼]

Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (alphabetaalpha) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features. [less ▲]

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See detailDesign of new delivery systems for therapeutic proteins based on calcium carbonate microspheres
Ramalapa, Bathabile ULiege; Crasson, Oscar ULiege; Vandevenne, Marylène ULiege et al

Poster (2015, December 10)

There exists a constant need for delivery systems that are biocompatible, offer bioactives protection from premature degradation and allow for targeted delivery and controlled release. Calcium carbonate ... [more ▼]

There exists a constant need for delivery systems that are biocompatible, offer bioactives protection from premature degradation and allow for targeted delivery and controlled release. Calcium carbonate (CaCO3) is one such system that has gained great favour for employment in the biomedical field due to possibilities of controlling size, morphology and crystalline forms of particles by tuning the synthesis conditions. CaCO3 has demonstrated ability to increase safety, stability and overall efficiency of protein therapeutics. The aim of the present work was to assess the significance of polysaccharide-protein complexes in enhancing the encapsulation of proteins in CaCO3 microspheres. A Chitin Binding Domain (ChBD), reported to have affinity for hyaluronic acid, was inserted on β-lactamase enzyme to develop a chimeric protein. The chimeric protein retained the activity of the enzyme and the binding properties and was encapsulated in CaCO3 microspheres by a super critical CO2 (ScCO2) process using hyaluronic acid as a templating agent. The particles were characterised in terms of size, zeta potential, morphology and protein loading. The results obtained confirmed the affinity of the ChBD to hyaluronic acid towards the production of stable, vaterite microparticles. Protein assays demonstrated that the ChBD enhanced the encapsulation of protein by up to 10 fold. Confocal images also suggested high encapsulation of the chimeric protein compared to native protein. Thus the production of polysaccharide-protein complexes seems effective in enhancing the encapsulation of proteins in CaCO3 microparticles using the ScCO2 process. Moreover this method will further be used to enhance encapsulation of therapeutic proteins such as growth factors for bone and cartilage regeneration. [less ▲]

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See detailThe N-terminal region of CHD4 is essential for activity and contains a HMG-box-like-domain that can bind poly(ADP-ribose).
Silva, Ana; Ryan, Daniel; Galanty, Y et al

in Journal of Biological Chemistry (2015)

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See detailEnzymatic functionalization of a nanobody using protein insertion technology
Crasson, Oscar ULiege; Rhazi, Noureddine; Jacquin, Olivier et al

in Protein Engineering, Design & Selection (2015), 28(10), 451-460

Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has be- come a ... [more ▼]

Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has be- come a major priority of biotech and pharmaceutical industries. Recently, a growing number of modified antibody-based products have emerged including fragments, multi-specific and conjugate antibodies. In this study, using protein engineering, we have functionalized the anti-hen egg-white lysozyme (HEWL) camelid VHH antibody fragment (cAb-Lys3), by insertion into a solvent-exposed loop of the Bacillus licheniformis β-lactamase BlaP. We showed that the generated hybrid protein conserved its enzymatic activity while the displayed nanobody retains its ability to inhibit HEWL with a nanomolar affinity range. Then, we successfully implemented the functionalized cAb-Lys3 in enzyme-linked immunosorbent assay, potentiometric biosensor and drug screening assays. The hybrid protein was also expressed on the surface of phage particles and, in this context, was able to interact specifically with HEWL while the β-lactamase activity was used to monitor phage interactions. Finally, using thrombin-cleavage sites surrounding the permissive insertion site in the β-lactamase, we reported an expression system in which the nanobody can be easily separated from its carrier protein. Altogether, our study shows that insertion into the BlaP β-lactamase consti- tutes a suitable technology to functionalize nanobodies and allowsthe creation of versatile tools that can be used in innovative biotechnological assays. [less ▲]

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See detailThe hidden face of the human macrophage chitotriosidase: taking a new look at this enzyme based on the biochemical and phylogenomic analysis of its chitin binding domain
Crasson, Oscar ULiege; Legrand, François; Léonard, Raphaël ULiege et al

Poster (2015, August)

Carbohydrates recognition is a critical process involved in numerous aspects of the cell biology such as inflammation, innate immune responses and proliferation. Chitin is an homopolysaccharide composed ... [more ▼]

Carbohydrates recognition is a critical process involved in numerous aspects of the cell biology such as inflammation, innate immune responses and proliferation. Chitin is an homopolysaccharide composed of β-1,4-linked N-acetylglucosamine (GlcNAc) units that is an abundant structural component of various infectious organisms like protozoans, nematodes and fungi. As there is no endogenous chitin produced by mammals, this polymer appeared to be a strategic target for innate immune agents which is why various carbohydrate binding proteins, associated or not with catalytic domains, are synthetized by plants and animals and are known to play a crucial role in innate immunity. The macrophage chitotriosidase (HCHT) is one of the three active chitinases synthetized by humans and has triggered significant attention recently due to its association with various inflammatory disorders. HCHT belongs to the Glycosyl Hydrolase family 18 (GH18) and is known to be involved in innate immunity. Nevertheless, its precise physiological function remains unclear. As numerous GHs, HCHT is a modular protein composed of a catalytic domain (GH18) associated to a Carbohydrate Binding Module (CBM) which is essential to hydrolyse crystalline chitin. If the catalytic domain GH18 is highly common in other GHs from animals, plants, fungi, bacteria, archea and viruses, its CBM (named ChBD) is much less conserved which makes the association between these two domains particularly intriguing. This work aims to demystify HCHT’s physiological function. Firstly, using competitive inhibition assays, we have highlighted the ability of ChBD to interact with chitooligosaccharides (GlcNAc1-2-4-6) which suggests that ChBD can potentially act as a lectin domain. Secondly, to better understand the molecular basis for chitin recognition, we have used homology modelling to build, with high confidence, the 3D structure model of ChBD. Based on this model, a specific set of residues has been selected for alanine scan mutagenesis which has allowed us to define the minimum chitin binding interface of the protein. Thirdly, Phylogenomic studies were performed to analyse the evolutionary history of the isolated catalytic and ChBD domains and understand how these domains were combined. Based on all these results, we discuss a new way of looking at HCHT where its ChBD would be the key determinant that has guided the catalytic domain from a basic metabolic function to a critical component of innate immunity in human. Finally, we propose a mechanism that explains how this enzyme could act at the molecular level to defend us against chitin-containing pathogens. [less ▲]

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See detailSite directed nitroxide spin labeling of oligonucleotides for NMR and EPR studies
Shepherd, Nicholas; Gamsjaeger, Roland; Vandevenne, Marylène ULiege et al

in Tetrahedron (2015), 71(5), 813-819

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See detailHow to build a biological linker dedicated to the engineering of novel drug delivery systems
Crasson, Oscar ULiege; Galleni, Moreno ULiege; Parente, Raffaella et al

Poster (2014, May 15)

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See detailEngineering Specificity Changes on a RanBP2 Zinc Finger that Binds Single-Stranded RNA.
Vandevenne, Marylène ULiege; O'Connell, M.R.; Helder, S. et al

in Angewandte Chemie International Edition (2014)

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