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

• Published in: Journal of agricultural and food chemistry 2017-09, Vol.65 (37), p.8154-8161
• Main Authors: Léger, Antoine, Hocquellet, Agnès, Dieryck, Wilfrid, Moine, Virginie, Marchal, Axel, Marullo, Philippe, Josseaume, Annabelle, Cabanne, Charlotte
• Format: Article
• Language: English
• Subjects: Chemical and Process Engineering; Chromatography, Ion Exchange; Engineering Sciences; Escherichia coli - genetics; Escherichia coli - metabolism; Food engineering; Gene Expression; Heat-Shock Proteins - genetics; Heat-Shock Proteins - isolation & purification; Heat-Shock Proteins - metabolism; Life Sciences; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - isolation & purification; Saccharomyces cerevisiae Proteins - metabolism
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/acs.jafc.7b02477

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.