Project R-2911

The development of an in vivo system for the site-specific modification of proteins with 'click' functionalized amino acids for biosensor applications (Research)

The immobilisation of proteins is of great importance for many biotechnological applications. A biosensor is a good example of such an application. A biosensor consists of three components: a biological receptor, a signal transducing platform and a data processor. The crucial part of a biosensor is the biological receptor. These receptors can bind very specific molecules, present in the solution to be analysed. When this binding event occurs, this results in a physical-chemical change, which can be turned into a measurable signal by the transducer. For an optimal and sensitive detection, it is necessary that the biomolecules are orientated on the surface with their active sites facing the target molecule. It is also very important that the coupling between the biomolecule and the surface is stable. To date, proteins are mainly coupled in a random way. This means that the active site of a fraction of the biomolecules is not available for their target, which leads to a reduced overall biological activity of the surface. For an optimal performance of a biosensor it is therefore important that the proteins are bound covalently and with a well-defined orientation. The strategies used so far cannot deliver this. To overcome the orientation and stability issues, an in vivo method will be developed to site-specifically incorporate bioorthogonal functional groups into proteins. Since there is a strong relationship between structure and function of proteins, and the protein conformation is strongly dependent on factors like pH and temperature, the reaction conditions used for the covalent coupling of proteins to substrates is of great influence. A coupling chemistry is needed that can be performed under mild conditions. A promising solution for this can be summarised under "click" chemistry. These reactions perform well in a physiological environment and room temperature and are therefore very useful for the coupling of proteins. A well-known type of "click" chemistry is the Huisgen 1,3-dipolar cycloaddition between alkynes and azides. The introduction of "click" chemistry into proteins will be done by using the 'amber suppression technique'. For this, a genetically encoded, mutant, orthogonal E.coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNACUA pair will be created and used for the expression of "click" modified proteins in yeast. A library of mutant EcTyrRS will be constructed and screened for selective incorporation of "click" modified amino acid. The benefit of this strategy is that it allows us to produce proteins that contain a genetically encoded orthogonal functional group (i.e. alkyne or azide) on a single, strategically chosen site in the protein. In this project, nanobodies will be used as a protein system. Incorporating a "click" modified amino acid on a genetically encoded position gives us the control to couple these proteins in a well-defined orientation. This makes it possible to produce surfaces that are homogonously covered and optimally bioactive. This project will be done in the research group: Organic and Biopolymere Chemistry (OBPC); where already one PhD-student and one post-doc are actively doing research in the site-specific modification of proteins and the covalent coupling of biomolecules to solid surfaces.

01 March 2011 - 28 February 2012