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Dominique Michaud – One of the best experts on this subject based on the ideXlab platform.

  • a protease activity depleted environment for heterologous proteins migrating towards the leaf cell apoplast
    Plant Biotechnology Journal, 2012
    Co-Authors: Charles Goulet, Moustafa Khalf, Frank Sainsbury, Marcandre Daoust, Dominique Michaud
    Abstract:

    Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then performed to test the efficiency of protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of Proteases from several families along the secretory pathway, including A1 and A22 Asp Proteases; C1A and C13 Cys Proteases; and S1, S8 and S10 Ser Proteases. In vitro protease assays confirmed the presence of various Proteases in N. benthamiana leaves, notably pointing to the deposition of A1- and S1-type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1/S1 protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 protease activities in this cell compartment, while increasing the leaf apoplast protein content by ~45% and improving the accumulation of a murine diagnostic antibody, C5-1, co-secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys Proteases, had no impact on the apoplast Proteases and protein content, but stabilized C5-1 in planta, presumably upstream in the secretory pathway. These data confirm, overall, the potential of protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway. © 2011 The Authors. Plant Biotechnology Journal

  • A protease activity–depleted environment for heterologous proteins migrating towards the leaf cell apoplast
    Plant Biotechnology Journal, 2011
    Co-Authors: Charles Goulet, Moustafa Khalf, Frank Sainsbury, Marc-andre D'aoust, Dominique Michaud
    Abstract:

    Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then performed to test the efficiency of protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of Proteases from several families along the secretory pathway, including A1 and A22 Asp Proteases; C1A and C13 Cys Proteases; and S1, S8 and S10 Ser Proteases. In vitro protease assays confirmed the presence of various Proteases in N. benthamiana leaves, notably pointing to the deposition of A1- and S1-type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1/S1 protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 protease activities in this cell compartment, while increasing the leaf apoplast protein content by ~45% and improving the accumulation of a murine diagnostic antibody, C5-1, co-secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys Proteases, had no impact on the apoplast Proteases and protein content, but stabilized C5-1 in planta, presumably upstream in the secretory pathway. These data confirm, overall, the potential of protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway. © 2011 The Authors. Plant Biotechnology Journal

  • Gel electrophoresis of proteolytic enzymes
    Analytica Chimica Acta, 1998
    Co-Authors: Dominique Michaud
    Abstract:

    This paper reviews the major procedures devised to efficiently detect and analyze proteolytic enzymes and their inhibitors after gel electrophoresis. After some considerations on the extraction of these proteins from biological samples and on the choice of appropriate substrates for the detection of protease activities, the major analytical schemes developed to detect electrophoretically separated Proteases are described. Given the well-known importance of protein protease inhibitors in the regulation of endogenous proteolytic enzymes and in host-pathogen and host-predator interactions, the detection of these proteins in crude extracts by the combined use of gel electrophoresis and specific Proteases is then considered. The use of gel electrophoresis in the study of protease/protease inhibitor complexes, useful in characterizing protease mechanistic classes and in monitoring the activity of recombinant proteinaceous inhibitors designed to regulate Proteases in various biological systems, is finally considered. Copyright (C) 1998 Elsevier Science B.V.

Olga Luisa Tavano – One of the best experts on this subject based on the ideXlab platform.

  • protein hydrolysis using Proteases an important tool for food biotechnology
    Journal of Molecular Catalysis B-enzymatic, 2013
    Co-Authors: Olga Luisa Tavano
    Abstract:

    Abstract This review intended to give a brief idea of the importance of Proteases applications. Processes that involve protein hydrolysis steps find wide ranging utilizations, such as cleaning process, proteomic studies, or food biotechnology process. Many positive effects hoped for with food processing can be achieved by protein hydrolysis using specific Proteases, changing nutritional, bioactive and functional properties of food proteins, which include improved digestibility, modifications of sensory quality (such as texture or taste), improvement of antioxidant capability or reduction in allergenic compounds. Protease applications in industrial processes are constantly being introduced and can be advantageous compared to chemical processes, by increasing hydrolysis specificity, product preservation and purity, and reducing environmental impact. Differences in specificity between Proteases are very important to take in to consideration as a guide for the choice of protease according to the protein source to be hydrolyzed or predicted products. In this present review, some aspects of the processes that involve protein hydrolysis steps are discussed, especially considering the application of specific Proteases as a tool on food biotechnology.

  • Protein hydrolysis using Proteases: An important tool for food biotechnology
    Journal of Molecular Catalysis B: Enzymatic, 2013
    Co-Authors: Olga Luisa Tavano
    Abstract:

    This review intended to give a brief idea of the importance of Proteases applications. Processes that involve protein hydrolysis steps find wide ranging utilizations, such as cleaning process, proteomic studies, or food biotechnology process. Many positive effects hoped for with food processing can be achieved by protein hydrolysis using specific Proteases, changing nutritional, bioactive and functional properties of food proteins, which include improved digestibility, modifications of sensory quality (such as texture or taste), improvement of antioxidant capability or reduction in allergenic compounds. Protease applications in industrial processes are constantly being introduced and can be advantageous compared to chemical processes, by increasing hydrolysis specificity, product preservation and purity, and reducing environmental impact. Differences in specificity between Proteases are very important to take in to consideration as a guide for the choice of protease according to the protein source to be hydrolyzed or predicted products. In this present review, some aspects of the processes that involve protein hydrolysis steps are discussed, especially considering the application of specific Proteases as a tool on food biotechnology. © 2012 Elsevier B.V. All rights reserved.

D I Pritchard – One of the best experts on this subject based on the ideXlab platform.

  • the partial characterization of Proteases present in the excretory secretory products and exsheathing fluid of the infective l3 larva of necator americanus
    International Journal for Parasitology, 1992
    Co-Authors: Sanjeev Kumar, D I Pritchard
    Abstract:

    Abstract Kumar S. and Pritchard D. I. 1992. The partial characterization of Proteases present in the excretory/secretory products and exsheathing fluid of the infective (L3) larva of Necator americanus . International Journal for Parasitology 22 : 563–572. Following the observation that live third-stage larvae (L3) could digest gelatin in vitro , gelatinolytic protease activity has been demonstrated at pH 8.5, in both exsheathing fluid (EF) and excretory/secretory (ES) products of infective L3 of Necator americanus . EF resolved as a single band of proteolytic activity, with a mol. wt of 116 kDa, while L3 ES products exhibited multiple bands of proteolysis, at 219, 200, 195, 166, 137, 92, 72 and 62 kDa; weak bands were detectable at 92 and 72 kDa. The EF protease was characterized as cysteine, whereas ES apparently possessed one serine (195 kDa) and seven (219, 200, 166, 137, 92, 72 and 62 kDa) cysteine protease bands and a combination of metallo- and cysteine Proteases of approximately the same mol. wts (62, 137 and 219 kDa). Though EF was not able to cleave immunoglobulins, ES was shown to cleave IgG, IgA and IgM, but not IgD or IgE. The activity appeared to be directed toward the Fc portion of the molecule, and was inhibited by PMSF, which is indicative of serine protease activity. The significance of the presence of such apparently diverse Proteases in larval products is discussed.

  • The partial characterization of Proteases present in the excretory/secretory products and exsheathing fluid of the infective (L3) larva of Necator americanus
    International Journal for Parasitology, 1992
    Co-Authors: Sanjeev Kumar, D I Pritchard
    Abstract:

    Abstract Kumar S. and Pritchard D. I. 1992. The partial characterization of Proteases present in the excretory/secretory products and exsheathing fluid of the infective (L3) larva of Necator americanus . International Journal for Parasitology 22 : 563–572. Following the observation that live third-stage larvae (L3) could digest gelatin in vitro , gelatinolytic protease activity has been demonstrated at pH 8.5, in both exsheathing fluid (EF) and excretory/secretory (ES) products of infective L3 of Necator americanus . EF resolved as a single band of proteolytic activity, with a mol. wt of 116 kDa, while L3 ES products exhibited multiple bands of proteolysis, at 219, 200, 195, 166, 137, 92, 72 and 62 kDa; weak bands were detectable at 92 and 72 kDa. The EF protease was characterized as cysteine, whereas ES apparently possessed one serine (195 kDa) and seven (219, 200, 166, 137, 92, 72 and 62 kDa) cysteine protease bands and a combination of metallo- and cysteine Proteases of approximately the same mol. wts (62, 137 and 219 kDa). Though EF was not able to cleave immunoglobulins, ES was shown to cleave IgG, IgA and IgM, but not IgD or IgE. The activity appeared to be directed toward the Fc portion of the molecule, and was inhibited by PMSF, which is indicative of serine protease activity. The significance of the presence of such apparently diverse Proteases in larval products is discussed.