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Title: Site selective labelling of proteins via native chemical ligation
Author: Rosenberg, Gillian
ISNI:       0000 0004 2682 1691
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2009
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A novel protein labelling strategy has been developed based on a chemical reaction known as Native Chemical Ligation (NCL). NCL is a highly specific and facile reaction, ligating a thioester specifically to an N-terminal cysteine residue and can be used to tag proteins in a selective and quantitative manner. This has advantages over traditional labelling methods such as use of green fluorescent protein: labelling with a small molecule is less likely to interfere with the protein’s native biological activity, and its physicochemical properties can be fine-tuned by synthesis. The key challenge is generating the protein bearing an N-terminal cysteine. The strategy developed in this thesis utilises the Foot-and-Mouth Disease Virus 3C protease (3Cpro), a highly selective protease that cleaves at PAKQ X with no apparent specificity for the P1’ position. To demonstrate the application of 3Cpro for NCL-mediated labelling, fluorescein was specifically ligated to the N-terminus of the PH domain of Protein Kinase B (PKB), which when defective is a key protagonist in a number of cancers. An expression–purification strategy was developed to express the protein as a fusion with a GST affinity tag, linked by the sequence PAKQC. The GST-PAKQC-PH protein was cleaved using 3Cpro and purified, yielding the N-terminal cysteine protein, C-PH, which was subsequently coupled to a fluorescein thioester via NCL. The labelled PH domain was successfully used as a probe of membrane bound phosphoinositol lipids in a fixed cell imaging assay. Suitable thioester tags are generated by converting an activated succinimide ester to a mercaptoethanesulfonate thioester using mercaptoethanesulfonate. This reaction was used to build up a labelling library of labels ranging from fluorescent dyes for imaging to biotin for pull-down studies, all of which were successfully ligated on the C-PH domain. This labelling strategy was then taken one step further to allow the specific labelling of the target from within a protein mixture. NCL is a generic method, requiring only a thioester and a cysteine N-terminus, therefore the versatility of this system can be exploited to create an easy to use labelling ‘toolbox’. The constructs and chemical tools produced here should enable facile production of a wide range of N-terminally labelled recombinant proteins for a vast array of studies.
Supervisor: Leatherbarrow, Robin ; Gaffney, Piers Sponsor: EPSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral