Methods and Reagents for Site-Specific Labeling of Peptides and Proteins

Brief Description

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.

Patent Information:
Category(s):
Research Tools
For Information, Contact:
Omar Bakht
University of Rochester
585.276.6600
omar_bakht@urmc.rochester.edu
Inventors:
Rudi Fasan
John Frost
Keywords: