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Neuhaus, Jean-Marc

Nom
Neuhaus, Jean-Marc
Affiliation principale
Fonction
Professeur ordinaire
Email
jean-marc.neuhaus@unine.ch
Identifiants
https://libra.unine.ch/handle/123456789/300

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  • Publication
    Métadonnées seulement
    Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium
    (2011)
    Sriranganadane, D.
    ;
    Reichard, U.
    ;
    Salamin, K.
    ;
    Fratti, M.
    ;
    Jousson, O.
    ;
    Waridel, P.
    ;
    Quadroni, M.
    ;
    Neuhaus, Jean-Marc 
    ;
    Monod, M.
    In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepDelta mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.
  • Publication
    Métadonnées seulement
    High-yield production and purification of recombinant T7-tag mature streptavidin in glucose-stressed E. coli
    (2008)
    Humbert, N.
    ;
    Schurmann, P.
    ;
    Zocchi, A.
    ;
    Neuhaus, Jean-Marc 
    ;
    Ward, T. R.
    The overexpression of toxic recombinant proteins is often problematic, leading to either low production levels or inclusion bodies. Streptavidin is no exception and thus the highest production level reported to date for streptavidin is 70 mg/L of functional protein. Herein, we report on the production in Escherichia coli and the purification of a recombinant mature streptavidin bearing a T7-tag. Optimization of critical parameters, including the glucose concentration, the pH and the time of induction as well as the use of BL21(DE3)pLysS cell strain, affords up to 120 mg/L functional streptavidin in soluble form. The yield can be further increased by an osmotic stress during the preculture by adding highly concentrated glucose before the inoculation of the culture medium, thus affording reproducibly 230 mg/L of soluble streptavidin. A single denaturing-renaturing step and affinity chromatography afford highly active tetrameric protein with >3.8/4.0 active sites.
  • Publication
    Accès libre
    A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole
    (1991)
    Neuhaus, Jean-Marc 
    ;
    Sticher, L.
    ;
    Meins, F. Jr.
    ;
    Boller, T.
    Tobacco contains different isoforms of chitinase (EC 3.2.1.14), a hydrolase thought to be involved in the defense against pathogens. Deduced amino acid sequences for putatively vacuolar, basic chitinases differ from the homologous extracellular, acidic isoforms by the presence of a C-terminal extension. To examine the role of this C-terminal extension in protein sorting, Nicotiana silvestris plants were stably transformed with chimeric genes coding for tobacco basic chitinase A with and without the seven C-terminal amino acids. In plants expressing unmodified chitinase A, the enzyme activity was low in the intercellular wash fluid but high in protoplasts and isolated vacuoles. In contrast, in plants expressing mutant chitinase lacking the C terminus, the activity was high in the intercellular wash fluid but low in protoplasts. N. silvestris plants were also transformed with similar constructions coding for a structurally unrelated, extracellular cucumber chitinase. In plants expressing unmodified cucumber chitinase, its activity was present in the intercellular wash fluid and absent from protoplasts. In plants expressing cucumber chitinase with the C-terminal extension from tobacco chitinase A, activity was low in intercellular wash fluids but high in protoplasts and vacuoles. These results demonstrate that the C-terminal extension of tobacco chitinase A is necessary and sufficient for the vacuolar localization of chitinases and, therefore, that it comprises a targeting signal for plant vacuoles.