It has become routine to generate proteins that will selectively bind to a targeted cell type. By attaching functional chemical components to these binding proteins, a variety of useful constructs could be generated. For example, attachment of a drug could enable delivery of the drug to the targeted cell type, or attachment of an imaging agent could enable selective visualisation of the cells in vivo. In order to facilitate attachment of multiple functional groups, we aimed to identify a simple and robust strategy for controlled synthesis of multifunctional constructs. The first strategy that we evaluated was attachment of multiple functional components to bromomaleimide linkers. Specifically, we tested a hypothesis that molecules attached to bromomaleimides could be released inside the targeted cell. This hypothesis was tested by generation of bromomaleimide-linked FRET pairs and microinjection of these into cells, which enabled release of components from bromomaleimides to be detected by fluorescence microscopy. We next evaluated a strategy in which multiple components are attached to apo neocarzinostatin (apo-NCS). It has been reported that Holo-NCS internalises into cells and releases its cargo in the nucleus, which raises the possibility that apo‑NCS could also deliver therapeutic ligands into cells. In order to test this possibility, apo-NCS was chemically labelled using rhodamine or biotin to detect internalisation. The final strategy that we evaluated was to react two cysteines on a single protein with dibromodiamide (DBDA). This reaction can generate a uniquely reactive group at each cysteine position, which can each be conjugated to a different functional chemical. We aimed firstly to demonstrate that this strategy could be applied to pairs of cysteines on other proteins. Secondly, we aimed to better understand the reaction of cysteine with DBDA. In an attempt to achieve both of these aims, DBDA was reacted with a library of single cysteine mutants.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:626820 |
| Date | January 2014 |
| Creators | Moody, P. R. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1420491/ |
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