Production and Purification of the Native Saccharomyces cerevisiae Hsp12 in Escherichia coli - INRAE - Institut national de recherche pour l’agriculture, l’alimentation et l’environnement
Article Dans Une Revue Journal of Agricultural and Food Chemistry Année : 2017

Production and Purification of the Native Saccharomyces cerevisiae Hsp12 in Escherichia coli

Résumé

Hsp12 is a small heat shock protein produced in many organisms, including the yeast Saccharomyces cerevisiae. It has been described as an indicator of yeast stress rate and has also been linked to the sweetness sensation of wine. To obtain a sufficient amount of protein, we produced and purified Hsp12 without tag in Escherichia coli. A simple fast two-step process was developed using a microplate approach and a design of experiments. A capture step on an anion-exchange salt-tolerant resin was followed by size exclusion chromatography for polishing, leading to a purity of 97%. Thereafter, specific anti-Hsp12 antibodies were obtained by rabbit immunization. An ELISA was developed to quantify Hsp12 in various strains of Saccharomyces cerevisiae. The antibodies showed high specificity and allowed the quantitation of Hsp12 in the yeast. The quantities of Hsp12 measured in the strains differed in direct proportion to the level of expression found in previous studies.
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Dates et versions

hal-02629235 , version 1 (27-05-2020)

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Antoine Léger, Agnès Hocquellet, Wilfrid Dieryck, Virginie Moine, Axel Marchal, et al.. Production and Purification of the Native Saccharomyces cerevisiae Hsp12 in Escherichia coli. Journal of Agricultural and Food Chemistry, 2017, 65 (37), pp.8154-8161. ⟨10.1021/acs.jafc.7b02477⟩. ⟨hal-02629235⟩

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