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.