Biocatalyst for Protein-Small Molecule Conjugation

Hedgehog terminal transferase (HTTase) provides a novel tool to covalently conjugate virtually any protein with a variety of small molecules, from fluorophores to therapeutics. HTTase is a water soluble protein that catalyzes protein conjugation in <60 min in the absence accessory proteins. Features of protein-conjugation with HTTase include: (1) two-component labeling kinetics (2) site-specific, stoichiometric modification (3) broad substrate tolerance and (4) labeling that is nearly traceless. The last feature is particularly noteworthy as existing protein conjugation methods require addition to the target protein of anywhere from 4 to 296 amino acids. That residual sequence can compromise function or engender an immune response during therapeutic application. Conjugation with HTTase requires addition to the target protein of a single glycine residue only.


There are similarities and key distinctions of HTTase compared with other biocatalysts for protein conjugation. Conjugation by HTTase is residue specific; it is active in physiological buffer at room temperature; the kinetics are relatively fast (half time, ~1 h or less); and a variety of protein substrates (substrate A) can be labeled with an increasing number of small molecules (substrate B). The closest analogues of HTTase seem to be the Sortase enzyme and the farnesyl transferase. There seem to be two key advantages of HTTase over these other biocatalysts for protein conjugation. First, HTTase allows one-pot, bi-molecular conjugation; whereas existing sortase and farnesyltransferase require 3 component reactions. Secondly, conjugation reaction with HTTase are nearly traceless, as the HTTase cleaves itself from the target protein during conjugation. The bi-molecular labeling (i) stems from the fact that the catalyst, HTTase, is fused to the protein substrate (substrate A), thus only the modifier (substrate B) needs to be added to the "pot" to initiate conjugation. No cofactors or accessory proteins are required. Existing methods that require at least three separate components (the protein of interest; the small molecule; and the conjugation catalyst). 3 component coupling reaction often proceed slowly, and require excess reactant concentration to drive conjugation to completion. The traceless feature (ii) arises from the fact that the protein of interest is liberated from HTTase upon labeling. Existing chemical labeling methods require addition to the protein of anywhere from 4 to 296 amino acids to allow recognition by the conjugation catalyst. That residual “scar” may compromise stability, perturb protein-protein association, or engender an immune response, which would be a serious concern for therapeutic applications. Thus, HTTase seems unrivaled in its stealth, leaving a post-labeling "scar" on the protein of interest of only single glycine residue.


Binghamton University RB479

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