Protein based molecular probes by unnatural amino acid incorporation

The "tag & modify" strategy for protein modification relies upon the genetic incorporation of an uncommon or unnatural amino acid into a protein backbone, followed by a chemo-selective modification to yield differentially modified proteins. This thesis describes the creation of a protein-based glycoconjugate tool for interrogating biological function. In Chapter 2, the unnatural amino acid, azidohomoalanine was genetically incorporated into a library of distance defined Np276 proteins via a selective pressure incorporation. Methods to prevent the common post translational modification N-terminal gluconylation were identified, including preliminary work on a small molecule intervention. The proteins were subsequently characterised with respect to other members of the (limited) family of pentapeptide repeat protein and the key biophysical parameters (TM, stability) with relate to it being a multivalent scaffold were investigated. In Chapter 3, An initial investigation into obtaining a selective amine modification initially via N-hydroxysuccinimide esters, led to the discovery (and characterisation) of a clear selectivity for N-terminal proline Isothiocyanate modification. The dual modification of proteins via the N-term Pro & the ubiquitous (glyco) copper(I)-catalysed azide alkyne cycloaddition was subsequently used to generate homogenously dual modified Np276 scaffolds. In Chapter 4, these proteins were then used in a FACS assay against a murine sialoadhesin - chinese hamster ovary cell line, the results showing promise for the further development of multivalent glycated probes of function.