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Neuhaus, Jean-Marc

Nom
Neuhaus, Jean-Marc
Affiliation principale
Fonction
Professeur.e émérite
Email
jean-marc.neuhaus@unine.ch
Identifiants
https://libra.unine.ch/handle/123456789/300
_
0000-0003-3288-101X

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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.