Léger Antoine, Hocquellet Agnès, Dieryck Wilfrid, Moine Virginie, Marchal Axel, Marullo Philippe, Josseaume Annabelle, Cabanne Charlotte
Bordeaux INP, CBMN, UMR 5248, F-33600 Pessac, France.
Biolaffort, 126 quai de la Souys, F-33100 Bordeaux, France.
J Agric Food Chem. 2017 Sep 20;65(37):8154-8161. doi: 10.1021/acs.jafc.7b02477. Epub 2017 Sep 11.
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.
热休克蛋白12（Hsp12）是一种在包括酿酒酵母在内的许多生物体中产生的小分子热休克蛋白。它被描述为酵母应激率的指标，并且还与葡萄酒的甜味感有关。为了获得足够量的蛋白质，我们在大肠杆菌中生产并纯化了无标签的Hsp12。采用微孔板方法和实验设计开发了一种简单快速的两步法。在耐阴离子交换盐树脂上进行捕获步骤，然后通过尺寸排阻色谱法进行纯化，纯度达到97%。此后，通过兔免疫获得了特异性抗Hsp12抗体。开发了一种酶联免疫吸附测定法（ELISA）来定量酿酒酵母各种菌株中的Hsp12。这些抗体显示出高特异性，并能够定量酵母中的Hsp12。在菌株中测得的Hsp12量与先前研究中发现的表达水平成正比。
