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

International Journal of Molecular Sciences

Yury Herasimenka¹  Marta Kotasinska¹  Stefan Walter¹ 

[1] FB Biology/Chemistry, Applied Genetics of Microorganisms, University Osnabrück, Barbarastr. 13, D-49069 Osnabrück, Germany;

关键词: chitin-assay;    chitin-binding protein;    chitin synthase;    electron microscopy;    polysaccharides;   

DOI  :  10.3390/ijms11093122

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.