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See detailIdentifying root-secreted proteases in Arabidopsis thaliana: an Activity-Based Protein Profiling approach.
Lallemand, Jérôme ULg; Périlleux, Claire ULg; Tocquin, Pierre ULg

Poster (2014, May 15)

Proteases are involved in many physiological processes during the whole life of the plant, such as embryonic development, defense against pathogens, nutrition or mycorrhiza creation. However, the ... [more ▼]

Proteases are involved in many physiological processes during the whole life of the plant, such as embryonic development, defense against pathogens, nutrition or mycorrhiza creation. However, the functions of many of the 800 proteases of Arabidopsis thaliana still remain unknown. Besides discovering new functions, studying proteases can also result in improving plant biotechnology. Indeed, plants can be used as hosts for recombinant protein production. Some proteins of interest require to be secreted in order to fold properly, but production yields are limited due to their degradation by endogenous extracellular proteases. The aim of our study is to identify active root-secreted proteases of Arabidopsis thaliana. Their activity was first analyzed by in vitro incubation with a target protein (BSA) at different values of pH and in the presence of proteases inhibitors. This analysis identified serine proteases as the major protease class involved in BSA degradation. Then, an Activity-Based Protein Profiling approach led to the labeling of two active serine proteases in the root-secreted sample. Finally, a further step towards the identification by mass spectrometry, based on affinity purification, was developed. [less ▲]

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See detailTowards the improvement of a rhizosecretion-based recombinant protein production system: Developing protease-depleted lines of Arabidopsis thaliana.
Lallemand, Jérôme ULg; Désiron, Carole ULg; Périlleux, Claire ULg et al

Poster (2013, June)

Besides traditional production systems, such as bacteria, yeasts and mammal cells, plants can now be used to produce eukaryotic recombinant proteins. Their advantages as hosts for protein production ... [more ▼]

Besides traditional production systems, such as bacteria, yeasts and mammal cells, plants can now be used to produce eukaryotic recombinant proteins. Their advantages as hosts for protein production include correct post-translational modifications, low cost of maintenance and no risk of contamination by human pathogens. Targeting heterologous proteins to the extracellular space is required for the correct folding of complex proteins and makes harvesting and purification easier. However, the quantity and the quality of recombinant proteins have been proved to be reduced by the action of endogenous co-secreted proteases. In this study, we aimed at identifying active root-secreted (rhizosecreted) proteases in the model plant Arabidopsis thaliana. Their activity was assayed by in vitro degradation of a target protein (Bovine Serum Albumine, BSA) in a range of pH. The protease classes involved in BSA degradation were evaluated by inhibitor-based assays that revealed serine proteases as the major class involved in this degradation in any tested conditions. As a first step towards identification, and subsequent silencing, of the most active members of this class, rhizosecreted proteases are being analyzed by the “Activity-Based Protein Profiling” approach. [less ▲]

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See detailTowards identification of active root-secreted proteases of Arabidopsis thaliana.
Lallemand, Jérôme ULg; Désiron, Carole ULg; Périlleux, Claire ULg et al

Poster (2013, April 18)

Besides traditional production systems, such as bacteria, yeasts and mammal cells, plants can now be used to produce eukaryotic recombinant proteins. Their advantages as hosts for proteins production ... [more ▼]

Besides traditional production systems, such as bacteria, yeasts and mammal cells, plants can now be used to produce eukaryotic recombinant proteins. Their advantages as hosts for proteins production include correct post-translational modifications, low cost of maintenance and no risk of contamination by human pathogens. Targeting heterologous proteins to the extracellular space is required for the correct folding of complex proteins and makes harvesting and purification easier. However, the quantity and the quality of recombinant proteins have been proved to be reduced by the action of endogenous co-secreted proteases. In this study, we characterized root-secreted proteases in the model plant Arabidopsis thaliana, at the activity and expression levels. Their activity was analyzed by in vitro degradation of a target protein (Bovine Serum Albumine, BSA) in a range of pH and in the presence of several proteases inhibitors. Serine proteases were identified as the major protease class involved in the degradation of BSA under all tested conditions. As a first step towards the identification of the key players, the expression level of selected members of this class was analyzed by quantitative RT-PCR in roots and leaves. [less ▲]

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See detailDegradation of recombinant IgG by root-secreted proteases of Arabidopsis thaliana and Nicotiana tabacum
Désiron, Carole ULg; Lallemand, Jérôme ULg; Périlleux, Claire ULg et al

Poster (2012, April 18)

Plants are promising hosts for the production of complex recombinant pharmaceuticals, such as antibodies (mAbs), because they offer an inexpensive and safer alternative to traditional production systems ... [more ▼]

Plants are promising hosts for the production of complex recombinant pharmaceuticals, such as antibodies (mAbs), because they offer an inexpensive and safer alternative to traditional production systems. The plant-based production of mAbs, which are multimeric glycoproteins, require their targeting to the secretory pathaway where they are properly folded and matured. However, co-secretion of endogenous proteases, which can represent up to 10% of the extracellular proteins (secretome), is known to significantly alter the yield and quality of secreted mAbs. In this study, we analyzed the proteolytic activities in root-secretome of Arabidopsis thaliana and Nicotiana tabacum. Root-secretomes were recovered by salt extraction and the protease activity was assayed in vitro or by zymography, in a range of pH. The relative contribution of protease classes was evaluated with specific inhibitors. [less ▲]

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