Step-By-Step In Vitro Mutagenesis: Lessons From Fucose-Binding Lectin PA-IIL
Language English Country United States Media print
Document type Journal Article
- Keywords
- Isothermal titration calorimetry (ITC), Lectins, Microscale thermophoresis, PA-IIL lectin, Protein crystallography, Site-directed mutagenesis method, Surface plasmon resonance,
- MeSH
- Bacterial Proteins genetics MeSH
- Fucose genetics MeSH
- Lectins genetics MeSH
- Mutagenesis, Site-Directed methods MeSH
- Mutagenesis genetics MeSH
- Pseudomonas aeruginosa genetics MeSH
- Protein Binding genetics MeSH
- Binding Sites genetics MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Bacterial Proteins MeSH
- fucose-binding lectin MeSH Browser
- Fucose MeSH
- Lectins MeSH
Site-directed mutagenesis is a powerful technique which is used to understand the basis of interactions between proteins and their binding partners, as well as to modify these interactions. Methods of rational design that are based on detailed knowledge of the structure of a protein of interest are often used for preliminary investigations of the possible outcomes which can result from the practical application of site-directed mutagenesis. Also, random mutagenesis can be used in tandem with site-directed mutagenesis for an examination of amino acid "hotspots."Lectins are sugar-binding proteins which, among other functions, mediate the recognition of host cells by a pathogen and its adhesion to the host cell surface. Hence, lectins and their binding properties are studied and engineered using site-directed mutagenesis.In this chapter, we describe a site-directed mutagenesis method used for investigating the sugar binding pattern of the PA-IIL lectin from the pathogenic bacterium Pseudomonas aeruginosa. Moreover, procedures for the production and purification of PA-IIL mutants are described, and several basic methods for characterizing the mutants are discussed.
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