Production, real time monitoring of recombinant human fibronectin domain's and their immobilization on bioactive surfaces : models or therapeutic purposes

Dans ce travail de thèse, nous avons développé un nouveau procédé de production et de purification en temps réel des domaines 9 et 10 de la fibronectine humaine chez E.coli. Cette stratégie combine trois partenaires de fusion en tandem : un double tag d’affinité (10xHis et SBP) et un tag de coloration (le domaine de fixation de l’hème du cytochrome b5) en présence d’un site Tev. Ce système a montré sa performance dans le traçage visuel des étapes d’expression et de purification et dans la quantification de la CMAT-FNIII9-10. La présence du double tag d’affinité permet une étape de purification simple et offre un degré de pureté atteignant les 98%. Par ailleurs, le cytochrome b5 a montré son intérêt dans le suivi visuel et quantitatif de l’adsorption de la CMAT-FNIII9-10 à la surface d’un support plastique. Ensuite, l’activité du fragment recombinant a été validée avec succès. Dans cette étude, nous avons construit une matrice adhésive en combinant les propriétés du polymère PCL avec celles de la CMAT-FNIII9-10. L’immobilisation de celle-ci s’est opérée d’une manière orientée en adsorbant la protéine de fusion à la surface des PCL par l’intermédiaire de la streptavidine. Cette approche a conduit à l’élaboration d’un matériau biofonctionnalisé en optimisant l’exposition des sites d’attachement cellulaire à la surface des PCL par une immobilisation orientée. La réponse cellulaire des CMS humaines a été validée efficacement sur cette matrice, en absence de sérum et en présence de BSA. Les résultats de cette expérience montrent que cette stratégie a contribué à améliorer l’exposition des sites « RGD » et « PHSRN » favorisant l’interaction avec les récepteurs cellulaires.In this thesis, we have developed a novel method for producing and purifying the 9th and 10thdomains of type III human fibronectin in E.coli. This strategy combines three fusion partnersin tandem: a dual affinity tag (10xHis and SBP) and a coloring tag (the binding domain of cytochrome b5 heme) in the presence of a Tev cleaving site.This system has demonstrated its performance in the visual tracking of the expression,purification and quantification steps of CMAT-FNIII9-10. The presence of the dual affinity tag allows a simple purification step and offers a degree of purity up to 98%. Moreover, the cytochrome b5 showed its interest in the visual and quantitative monitoring of the CMATFNIII9-10 adsorption onto the surface of a plastic support. Then the biological activity of there combinant fragment was successfully validated. In this study, we constructed an adhesive matrix by combining the properties of PCL with those of CMAT-FNIII9-10. Immobilization of the recombinant fragments is carried out by an oriented adsorption of the fusion protein onto the PCL film through a streptavidin layer. This approach has led to the development of a bio-functionalized material by optimizing the exposure of cell attachment sites on the surface of the PCL by an oriented immobilization. The cellular response of human MSCs was effectively validated on this matrix, in the absence of serum and in the presence of BSA. The results of this experiment show that this strategy has helped to improve the exposure sites "RGD" and "PHSRN" promoting interaction with cellular receptors

Production, traçage en temps réel de domaines de la fibronectine humaine recombinante et immobilisation des recombinants sur des surfaces bioactives : modèles ou à visées thérapeutiques

In this thesis, we have developed a novel method for producing and purifying the 9th and 10thdomains of type III human fibronectin in E.coli. This strategy combines three fusion partnersin tandem: a dual affinity tag (10xHis and SBP) and a coloring tag (the binding domain of cytochrome b5 heme) in the presence of a Tev cleaving site.This system has demonstrated its performance in the visual tracking of the expression,purification and quantification steps of CMAT-FNIII9-10. The presence of the dual affinity tag allows a simple purification step and offers a degree of purity up to 98%. Moreover, the cytochrome b5 showed its interest in the visual and quantitative monitoring of the CMATFNIII9-10 adsorption onto the surface of a plastic support. Then the biological activity of there combinant fragment was successfully validated. In this study, we constructed an adhesive matrix by combining the properties of PCL with those of CMAT-FNIII9-10. Immobilization of the recombinant fragments is carried out by an oriented adsorption of the fusion protein onto the PCL film through a streptavidin layer. This approach has led to the development of a bio-functionalized material by optimizing the exposure of cell attachment sites on the surface of the PCL by an oriented immobilization. The cellular response of human MSCs was effectively validated on this matrix, in the absence of serum and in the presence of BSA. The results of this experiment show that this strategy has helped to improve the exposure sites "RGD" and "PHSRN" promoting interaction with cellular receptors.Dans ce travail de thèse, nous avons développé un nouveau procédé de production et de purification en temps réel des domaines 9 et 10 de la fibronectine humaine chez E.coli. Cette stratégie combine trois partenaires de fusion en tandem : un double tag d’affinité (10xHis et SBP) et un tag de coloration (le domaine de fixation de l’hème du cytochrome b5) en présence d’un site Tev. Ce système a montré sa performance dans le traçage visuel des étapes d’expression et de purification et dans la quantification de la CMAT-FNIII9-10. La présence du double tag d’affinité permet une étape de purification simple et offre un degré de pureté atteignant les 98%. Par ailleurs, le cytochrome b5 a montré son intérêt dans le suivi visuel et quantitatif de l’adsorption de la CMAT-FNIII9-10 à la surface d’un support plastique. Ensuite, l’activité du fragment recombinant a été validée avec succès. Dans cette étude, nous avons construit une matrice adhésive en combinant les propriétés du polymère PCL avec celles de la CMAT-FNIII9-10. L’immobilisation de celle-ci s’est opérée d’une manière orientée en adsorbant la protéine de fusion à la surface des PCL par l’intermédiaire de la streptavidine. Cette approche a conduit à l’élaboration d’un matériau biofonctionnalisé en optimisant l’exposition des sites d’attachement cellulaire à la surface des PCL par une immobilisation orientée. La réponse cellulaire des CMS humaines a été validée efficacement sur cette matrice, en absence de sérum et en présence de BSA. Les résultats de cette expérience montrent que cette stratégie a contribué à améliorer l’exposition des sites « RGD » et « PHSRN » favorisant l’interaction avec les récepteurs cellulaires

A new supramolecular chromium(III) complex: Synthesis, structural determination, optical study, magnetic and antibacterial activity

International audienc

A new tagged-TEV protease: Construction, optimisation of production, purification and test activity

International audienc

Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption

Engineering of biomimetic motives have emerged as promising approaches to improving cells’ binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface’s receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5β1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications

WITHDRAWN: Reprint of: A new tagged-TEV protease: Construction, optimisation of production, purification and test activity

International audienc

An Improved Strategy for Easy Process Monitoring and Advanced Purification of Recombinant Proteins

International audienc

An Improved Strategy for Easy Process Monitoring and Advanced Purification of Recombinant Proteins

International audienc

Value of inferior vena cava collapsibility index as marker of heart failure in chronic obstructive pulmonary disease exacerbation

Abstract Introduction Inferior vena cava (IVC) diameter variability with respiration measured by ultrasound was found to be useful for the diagnosis of heart failure (HF) in ED patients with acute dyspnea. Its value in identifying HF in acute exacerbation of chronic obstructive pulmonary disease exacerbation (AECOPD) was not specifically demonstrated. Objective To determine the value of ΔIVC in the diagnosis of HF patients with AECOPD. Methods This is a prospective study conducted in the ED of three Tunisian university hospitals including patients with AECOPD. During this period, 401 patients met the inclusion criteria. The final diagnosis of HF is based on the opinion of two emergency experts after consulting the data from clinical examination, cardiac echocardiography, and BNP level. The ΔIVC was calculated by two experienced emergency physicians who were blinded from the patient’s clinical and laboratory data. A cut off of 15% was used to define the presence (< 15%) or absence of HF (≥ 15%). Left ventricular ejection fraction (LVEF) was also measured. The area under the ROC curve, sensitivity, specificity, and positive and negative predictive values were calculated to determine the diagnostic and predictive accuracy of the ΔIVC in predicting HF. Results The study population included 401 patients with AECOPD, mean age 67.2 years with male (68.9%) predominance. HF was diagnosed in 165 (41.1%) patients (HF group) and in 236 patients (58.9%) HF was excluded (non HF group). The assessment of the performance of the ΔIVC in the diagnosis of HF showed a sensitivity of 37.4% and a specificity of 89.7% using the threshold of 15%. The positive predictive value was 70.9% and the negative predictive value was 66.7%. The area under the ROC curve was 0.71(95%, CI 0.65–0.76). ΔIVC values were not different between HF patients with reduced LVEF and those with preserved LVEF. Conclusion Our results showed that ΔIVC has a good value for ruling out HF in ED patients consulting for AECOPD