Christopher Allan
Phone:  +44 131 650 7546
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Address Joseph Black Building
School of Chemistry
David Brewster road
Edinburgh
EH9 3FJ
United Kingdom

 

Project title: Targeting Tyrosine: a catch and release approach to protein-tagging

Project outline:
The development of modern super-resolution imaging techniques allow observation of cellular processes on the nm scale. With these advances come the requirement for bespoke fluorescent probes which are labelled with organic fluorophores in a 1:1 stoichiometry. In this project, a novel method of protein-tagging has been developed which will be used to produce fluorescent nanobodies for use in super-resolution imaging studies. This method targets a native amino acid residue and utilises the streptavidin-biotin binding complex in a solid phase catch and release system. For its relatively low abundance and selective nucleophilicity, tyrosine (Tyr) was selected as the ideal native target, allowing this method to be applied to native-state biomolecules without prior incorporation of a non-canonical amino acid. The solid phase platform was developed to favour mono-conjugation and allow straightforward purification. Formation of a chemically-labile diazo linker was used to immobilise the Tyr-bearing biomolecule on a solid support. Following purification by solid-phase extraction, the linker was cleaved under mild reducing conditions to furnish the modified biomolecule. The unique aromatic amine of the cleavage product is then further functionalised for conjugation to the desired fluorophore. Once the fluorophore is developed it will be tested using the appropriate super-resolution technique.

ChrisA

 

Conferences:

SULSA Research Symposium 2014
ISACS Challenges in Chemical Biology 2015
ESRIC Super-Resolution Summer School 2015
ESRIC Interdisciplinary Consortium Meeting 2015 
RSC Chemical Biology & Bio-organic Group Postgraduate Symposium 2016
TransMed Student Conference 2016