Novel Binding Specificities Engineered into the Scaffold of a Carbohydrate Binding Module

The growing field of biotechnology is in constant need of proteins that can function as recognition tools for separational, analytical and therapeutic purposes. Different molecular engineering approaches are applied on natural proteins in order to create variants with desired properties. This thesis is based on five original papers that deal with selection, characterisation and application of novel binding specificities engineered into the scaffold of a carbohydrate binding module that originates from a xylanase in the thermophilic bacterium Rhodothermus marinus. Molecular evolution studies on this scaffold allowed for the generation of variants that bind specifically to the carbohydrate targets xylan, AvicelTM, mannan and xyloglucan. In addition, the scaffold employed in this work was also able to adopt specific protein recognition to a human IgG4 molecule. Apart from high binding specificities, the engineered proteins have additional properties such as high thermal stability and ease of production in Escherichia coli, which are advantageous in most applications. One of the papers in this thesis demonstrates the potential use of the created xylanbinding variants for detection of their target in wood fibres and plant sections. Also, generated variants with other binding specificities have the potential to find similar use as bioanalytical tools. In conclusion, the scaffold of the carbohydrate binding module engaged in the engineering studies of this thesis proved to be suitable for carrying diversity and has thus allowed for the creation of novel variants with diverse binding specificities useful in biotechnological applications.