Brief
Description:
Methods
for site-specific protein functionalization find a broad range of applications
in the context of protein detection, imaging, and purification, in the
development of improved protein-based therapeutics (e.g. via PEGylation), and in
the preparation of protein microarrays. This invention describes a new chemical
method for site-specifically labeling a target protein at its C-terminus with an
organic molecule of interest, such as a fluorescent dye, an affinity tag, a
polymer, a drug molecule, etc. This methodology involves the use of novel
reagents that can efficiently react with an intein-fused protein and form a
stable covalent bond with the C-terminal end of the target protein, while
excising the intein moiety. This methodology can be applied for the efficient,
chemo- and site-selective C-terminal functionalization of a protein with a
variety of organic molecules (e.g. fluorescent dye, biotin, polymer, etc.) under
mild reaction conditions. The labeling procedure can be carried out in vitro, in
complex media (e.g. cell lysate) and, in the case of cell-permeable cargo
molecules, also in vivo (i.e. target protein inside a living cell).
Advantages:
Currently
available methods for chemoselective C-terminal labeling of proteins rely on
so-called expressed protein ligation techniques, which involve the reaction of
cysteine-based reagents with intein-fusion proteins. These procedures, however,
suffer from low ligation efficiencies, slow reaction rates, and the requirement
for exogenous catalysts (i.e thiols), which limits the scope of these labeling
methods in particular when rapid detection, high-yield labeling, and in vivo
applicability are desired. The methodologies of this invention enable to
overcome these limitations, eliminating the need for exogenous catalysts and
granting faster reaction rates, higher yields of labeled protein, and the
possibility to site-selectively label proteins in living cells.