Herasimenka Yury, Kotasinska Marta, Walter Stefan, Schrempf Hildgund
FB Biology/Chemistry, Applied Genetics of Microorganisms, University Osnabrück, Barbarastr. 13, D-49069 Osnabrück, Germany; E-Mails:
Int J Mol Sci. 2010 Sep 7;11(9):3122-37. doi: 10.3390/ijms11093122.
A new assay system for chitin has been developed. It comprises the chitin-binding protein ChbB in fusion with a His-tag as well as with a Strep-tag, the latter of which was chemically coupled to horseradish peroxidase. With the resulting complex, minimal quantities of chitin are photometrically detectable. In addition, the assay allows rapid scoring of the activity of chitin-synthases. As a result, a refined procedure for the rapid purification of yeast chitosomes (nano-machineries for chitin biosynthesis) has been established. Immuno-electronmicroscopical studies of purified chitosomes, gained from a yeast strain carrying a chitin-synthase gene fused to that for GFP (green-fluorescence protein), has led to the in situ localization of chitin-synthase-GFP molecules within chitosomes.
已开发出一种新的几丁质检测系统。它由与His标签以及Strep标签融合的几丁质结合蛋白ChbB组成，其中Strep标签化学偶联到辣根过氧化物酶上。利用所得复合物，可通过光度法检测到极少量的几丁质。此外，该检测方法还能快速测定几丁质合酶的活性。因此，已建立了一种用于快速纯化酵母几丁质体（几丁质生物合成的纳米机器）的精细程序。对从携带与绿色荧光蛋白基因融合的几丁质合酶基因的酵母菌株中获得的纯化几丁质体进行免疫电子显微镜研究，已实现几丁质合酶-绿色荧光蛋白分子在几丁质体内的原位定位。
