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Int. J. Mol. Sci. 2010, 11(9), 3122-3137; doi:10.3390/ijms11093122

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

FB Biology/Chemistry, Applied Genetics of Microorganisms, University Osnabrück, Barbarastr. 13, D-49069 Osnabrück, Germany
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Received: 22 June 2010 / Revised: 14 August 2010 / Accepted: 26 August 2010 / Published: 7 September 2010
(This article belongs to the Special Issue Chitins)
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Abstract

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. View Full-Text
Keywords: chitin-assay; chitin-binding protein; chitin synthase; electron microscopy; polysaccharides chitin-assay; chitin-binding protein; chitin synthase; electron microscopy; polysaccharides
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Herasimenka, Y.; Kotasinska, M.; Walter, S.; Schrempf, H. A Selective Assay to Detect Chitin and Biologically Active Nano-Machineries for Chitin-Biosynthesis with Their Intrinsic Chitin-Synthase Molecules. Int. J. Mol. Sci. 2010, 11, 3122-3137.

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