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

Yury Herasimenka,Hildgund Schrempf,Marta Kotasinska,Stefan Walter

doi:10.3390/ijms11093122

Yury Herasimenka, Hildgund Schrempf + Show 2 more

Open Access

https://doi.org/10.3390/ijms11093122

Copy DOI

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.

Highlights

The chitin recognition proteins (CHBs) from streptomycetes are small (18–19 kDa) secreted proteins, which either highly recognize α chitin (i.e., CHB1, CHB2) or are less selective, Int
The presented test system relies on the chitin-binding protein ChbB carrying either a His-tag or a
The ChbB-Horseradish peroxidase (HRP) complexes proved to be considerably more specific for nascent chitin than the previously described wheat-germ agglutinin (WGA)-HRP adduct [21] or an assay depending on radioactively labeled substrate [25]

Summary

Introduction

The chitin recognition proteins (CHBs) from streptomycetes are small (18–19 kDa) secreted proteins, which either highly recognize α chitin (i.e., CHB1, CHB2) or are less selective, Int. J. 2010, 11 such as CHB3 targeting α and β chitin, as well as chitosan, the deacetylated chitin-derivative [1,2,3,4,5]. Based on the findings for streptomycetes, we identified the homologous chitin-binding protein ChbB) from Bacillus amyloliquefaciens ALKO 2718, recognizing α and β chitin, but not chitosan [6]. We obtained the corresponding protein (after overexpression of the chbB gene in E. coli in frame with six histidine codons) as a homogeneous type in larger quantities, than the Streptomyces CHB1-type from its natural host or after its overexpression in E. coli [7,8]

Objectives

Results

Conclusion