title: Fusion of a novel genetically engineered chitosan affinity protein and green fluorescent protein for specific detection of chitosan in vitro and in situ.
abstract: Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan-e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His(6) tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His(5) antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining.
authors.affiliation: Institute of Plant Biology and Biotechnology, University of Münster, Münster, Germany
authors.forename: Malathi
authors.initials: M
authors.lastname: Nampally
authors.forename: Bruno Maria
authors.initials: BM
authors.lastname: Moerschbacher
authors.forename: Stephan
authors.initials: S
authors.lastname: Kolkenbrock
mesh_terms: D019677:Artificial Gene Fusion; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D048271:Chitosan / Q000032:analysis*; D060026:Endophytes / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005658:Fungi / Q000302:isolation & purification; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D013194:Staining and Labeling / Q000379:methods*; D014908:Triticum / Q000382:microbiology
chemical_list: D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase