Methods for protein ligation and uses thereof
Inventors
Squire, Christopher John • BAKER, Edward Neill • YOUNG, Paul Gary • YOSAATMADJA, Yuliana
Assignees
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Publication Number
US-11807671-B2
Publication Date
2023-11-07
Expiration Date
Abstract
The invention relates to protein ligation technologies, purified or recombinant peptides, methods for making peptides and proteins with covalent bonds including reversible covalent bonds such as reversible intermolecular covalent bonds, and uses thereof. In particular, this invention relates to intermolecular ester bonds, particularly reversible ester bonds between the hydroxyl and amide groups of amino acid side chains present in recombinant chimeric peptides and proteins and the use of such peptides and proteins in protein engineering, for example in the preparation of multimeric protein complexes, including functionalised multimeric protein complexes.
Core Innovation
The invention relates to a peptide tag and a binding partner pair in which each component comprises a separate fragment of an Ig-like fold of a Cpe-like (β-clasp) domain, with one or more reactive residues positioned so that the two fragments spontaneously form an intermolecular ester bond. The peptide tag comprises at least about 10 contiguous amino acids of an Ig-like fold of a Cpe-like domain in a β-clasp from a β-clasp containing protein, and the peptide tag does not comprise the entire amino acid sequence of the β-clasp containing protein.
The binding partner comprises a separate fragment that also contains at least about 10 contiguous amino acids of a complementary part of the Ig-like fold. The peptide tag and binding partner are covalently bound to each other only by the intermolecular ester bond between the reactive residues, and one or both are covalently linked to one or more heterologous amino acid sequences.
The intermolecular ester bond is formed covalently only between specified reactive residues, with either serine or threonine on one partner and glutamine or glutamate/glutamic acid on the other, or the complementary arrangement with glutamine/glutamate on the peptide tag side. The disclosure further describes reversible behavior of the covalent intermolecular ester bond, including controlling ester formation versus hydrolysis.
Claims Coverage
The patent includes one independent claim defining a covalent peptide-tag/binding-partner pair that forms only a specified intermolecular ester bond between reactive residues in Ig-like Cpe-like (β-clasp) domain fragments; the dependent claims further refine the reactive residues, reversibility/hydrolysis behavior, structural/proximity constraints, optional sequence sources/regions, and heterologous payload types.
Ig-like Cpe-like β-clasp fragments forming only an intermolecular ester bond
A peptide tag and a separate binding partner fragment, each comprising at least about 10 contiguous amino acids of an Ig-like fold of a Cpe-like (β-clasp) domain from a β-clasp containing protein, where the peptide tag and binding partner are covalently bound only by an intermolecular ester bond between reactive residues, with the peptide tag not comprising the entire amino acid sequence of the β-clasp containing protein.
Specified Ser/Thr versus Gln/Glu/glutamic acid reactive residue pairing
Either the reactive residue in the peptide tag is serine or threonine while the reactive residue in the binding partner is glutamine or glutamate/glutamic acid, or vice versa, such that the intermolecular ester bond is formed between the reactive residues in the two partners.
Covalent linking to heterologous amino acid sequences
The peptide tag or the binding partner or both are covalently linked to one or more heterologous amino acid sequences.
Claim coverage centers on a β-clasp Ig-like fold peptide-tag/binding-partner system that covalently pairs only through a reactive-residue-directed intermolecular ester bond, with the Ser/Thr–Gln/Glu (or reciprocal) pairing requirement and optional covalent linkage of heterologous amino acid sequences.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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