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

Autor(en): Herasimenka, Yury
Kotasinska, Marta
Walter, Stefan
Schrempf, Hildgund 
Stichwörter: ALPHA-CHITIN; BINDING PROTEIN CHB1; Biochemistry & Molecular Biology; Chemistry; Chemistry, Multidisciplinary; chitin synthase; chitin-assay; chitin-binding protein; electron microscopy; EVOLUTION; LOCALIZATION; polysaccharides; SACCHAROMYCES-CEREVISIAE; STREPTOMYCES-OLIVACEOVIRIDIS
Erscheinungsdatum: 2010
Herausgeber: MDPI
Volumen: 11
Ausgabe: 9
Startseite: 3122
Seitenende: 3137
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.
DOI: 10.3390/ijms11093122

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