A Selective Assay to Detect Chitin and Biologically Active Nano-Machineries for Chitin-Biosynthesis with Their Intrinsic Chitin-Synthase Molecules

作者：Herasimenka Yury; Kotasinska Marta; Walter Stefan; Schrempf Hildgund^*

来源：International Journal of Molecular Sciences, 2010, 11(9): 3122-3137.

DOI：10.3390/ijms11093122

• chitin-assay
• chitin-binding protein
• chitin synthase
• electron microscopy
• polysaccharides

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.

• 出版日期2010-9