CN-122029288-A - Method for transpeptidation

Abstract

The present invention provides methods of transpeptidation, transpeptidation products obtainable from the methods, and chimeric fusion polypeptides. An exemplary method of transpeptidation includes ligating a donor substrate with an acceptor protein or peptide using a transpeptidase, wherein the donor substrate is a protein, peptide, amino acid amide or amino acid ester, and wherein the ligating reaction produces a ligation product and a byproduct that is a substrate for the transpeptidase, and cleaving one or more amino acids from the end of the byproduct using an exopeptidase such that the truncated byproduct is not a substrate for the transpeptidase, thereby shifting the reaction balance of the transpeptidation reaction toward the ligation product.

Inventors

• Michael. Webb
• Bruce. Turnbull
• Christian Hollingworth
• Zoe Arnot

Assignees

• 利兹大学

Dates

Publication Date

20260512

Application Date

20240910

Priority Date

20230911

Claims (20)

1. 1. A method of transpeptidation comprising: Ligating a donor substrate with an acceptor protein or peptide using a transpeptidase, wherein the donor substrate is a protein, peptide, amino acid amide or amino acid ester, and wherein the ligating reaction produces a ligation product and a byproduct that is a substrate for the transpeptidase, Cleavage of one or more amino acids from the end of the byproduct using an exopeptidase, such that the truncated byproduct is not a substrate for the transpeptidase, shifts the reaction equilibrium of the transpeptidation reaction toward the ligation product.
2. 2. The method of claim 1, wherein the exopeptidase is an aminopeptidase, and wherein the one or more amino acids are cleaved from the N-terminus of the byproduct.
3. 3. The method of claim 2, wherein the aminopeptidase is a D-aminopeptidase.
4. 4. A method according to claim 2 or claim 3, wherein the aminopeptidase is a glycyl aminopeptidase.
5. 5. A method according to claim 3, wherein the D-aminopeptidase is human brucella (Brucella anthropi) D-aminopeptidase.
6. 6. The method of any one of claims 2 to 5, wherein the aminopeptidase is sequence-specific, optionally wherein the aminopeptidase is specific for a GX 2 -terminated peptide, wherein X 2 is a small, polar or negatively charged amino acid, or other glycinamide derivative.
7. 7. The method of any preceding claim, wherein the N-terminus of the donor substrate comprises a motif that is not a substrate for the exopeptidase.
8. 8. The method of claim 7, wherein the N-terminus of the donor substrate comprises motif XX 1 , wherein X 1 is a hydrophobic or positively charged amino acid.
9. 9. The method of claim 8, wherein the N-terminus of the donor substrate comprises motif GX 1 , wherein X 1 is a hydrophobic or positively charged amino acid, optionally wherein the N-terminus of the donor substrate comprises motif GV.
10. 10. The method of any preceding claim, wherein the receptor protein or peptide comprises a recognition motif that is cleaved by the transpeptidase to produce the byproduct.
11. 11. The method of claim 10, wherein the recognition motif comprises LPXT/G or LPXT/GX 2 such that the byproduct comprises glycinamide or GX 2 , optionally wherein the recognition motif comprises LPET/G or LPET/GX 2 .
12. 12. The method of claim 11, wherein X 2 is a small, polar, or negatively charged amino acid, optionally wherein X 2 is a.
13. 13. The method of any preceding claim, wherein the N-terminus of the donor substrate comprises a motif that is not a substrate for the exopeptidase, and wherein the acceptor protein or peptide comprises a recognition motif that is cleaved by the transpeptidase to produce the byproduct.
14. 14. The method of claim 13, wherein the N-terminus of the donor substrate comprises motif GX 1 , wherein X 1 is a hydrophobic or positively charged amino acid, and wherein: (i) The receptor protein or peptide comprises the motif X/GX 2 , wherein/is the cleavage site of the transpeptidase and X 2 is a small, polar or negatively charged amino acid, or (Ii) The receptor protein or peptide comprises the motif X/G-amide, wherein the amide group is-CONH 2 、-CONR 2 or-CONHR.
15. 15. The method of claim 14, wherein the N-terminus of the donor substrate comprises motif GX 1 , wherein X 1 is a hydrophobic or positively charged amino acid, and wherein the acceptor protein or peptide comprises the motif LPXT/GX 2 , wherein/is the cleavage site of the transpeptidase, and X 2 is a small, polar or negatively charged amino acid or amide group.
16. 16. The method of any preceding claim, wherein the transpeptidase is a cysteine transpeptidase.
17. 17. The method of any preceding claim, wherein the transpeptidase is a sortase, butelase, or an asparaginyl endopeptidase.
18. 18. The method of claim 17, wherein the transpeptidase is a sortase.
19. 19. The method of claim 18, wherein the transpeptidase is sortase a, optionally wherein the sortase a is streptococcus pyogenes (Streptococcus pyogenes) sortase a or an evolved form thereof.
20. 20. The method of any preceding claim, wherein the transpeptidase and the exopeptidase are provided by a chimeric fusion polypeptide.

Description

Method for transpeptidation Technical Field The present invention relates to a method of transpeptidation. Background Site-specific labelling of proteins is a widely used tool in both industrial and academic research. Transpeptidases, such as sortase (sortase), butelase, and asparaginyl endopeptidase (ASPARAGINYL ENDOPEPTIDASE), provide an effective strategy for labeling the N-and C-termini of proteins. This approach has now been developed and applied to an increasing number of complex systems, including cell surface labeling, in vivo labeling and the generation of therapeutic antibody-drug conjugates. Many improvements to these methods have been reported, including evolution of enzymes with enhanced and altered reactivity, use of multiple enzymes to achieve orthogonal labeling reactions, and methods of developing labeled internal residues. However, in almost all cases, it is necessary to over-dose one of the reaction components to achieve complete conversion of unlabeled protein to labeled protein. Transpeptidase recognizes a defined peptide motif in its substrate protein or peptide. The active site cysteine residue reacts with the motif to form a thioacyl intermediate, which is then reacted with a substrate peptide or protein to form a product. A key challenge in such labelling reactions is that they are typically fully reversible (fig. 1A). The substrate recognition motif of the enzyme (e.g., LPXT/G for sortase) is still present in the product of the reaction, and the byproducts generated during initial thioacyl intermediate formation are also substrates for the second step. The reversibility of the reaction results in its thermodynamic control, which results in an equilibrium mixture of labeled and unlabeled products (fig. 1B). The production of pure labeled proteins typically requires a large excess of labeled peptide and/or removal of unlabeled protein from the reaction mixture. For N-terminal labeling, the use of an ester/depsipeptide (DEPSIPEPTIDE) substrate has been reported, wherein the by-product alcohol is no longer a substrate for the enzyme (fig. 1C). However, this strategy is not applicable to C-terminal labeling because it requires the incorporation of an ester linkage in the expressed protein. Thus, a number of alternative strategies have been developed for C-terminal tagging. For example, by using a centrifugal filtration device (5), the by-product peptide is physically removed for C-terminal labeling using a chemical reagent to react with the product peptide (6) or using high concentration Ni 2+ to chelate (7, 8). In other work, substrate preference of asparagine endopeptidases has been exploited to achieve selective reactions (9). However, none of these previous strategies allow one-pot controlled quantitative peptide ligation using a transpeptidase that shifts the reaction equilibrium towards the ligation product and is applicable to both N-terminal and C-terminal tags. Disclosure of Invention In a first aspect, a method of transpeptidation is provided comprising ligating a donor substrate with an acceptor protein or peptide using a transpeptidase, wherein the donor substrate is a protein, peptide, amino acid amide or amino acid ester, and wherein the ligating reaction produces a ligation product and a byproduct that is a substrate for the transpeptidase, and then cleaving one or more amino acids from the end of the byproduct using an exopeptidase such that the truncated byproduct is not a substrate for the transpeptidase, thereby shifting the reaction balance of the transpeptidation reaction towards the ligation product. The present inventors developed a coupled enzymatic transpeptidation system comprising a combination of transpeptidases and exopeptidases. Advantageously, the by-products are degraded by the exopeptidase, thereby removing the by-products which are substrates for the transpeptidase. Thus, the reaction equilibrium is shifted forward, facilitating ligation of the products and preventing reversibility of the reaction. The method greatly enhances the efficiency of the transpeptidation reaction and promotes the wider application of the method in experimental and industrial processes. In addition, coupled enzymatic transpeptidation systems achieve both quantitative N-and C-terminal labeling of proteins or peptides and quantitative fusion of expressed peptides or proteins. Methods of transpeptidation are suitable for a variety of applications including, but not limited to, protein or peptide labeling, conjugation of a protein or peptide to a therapeutic agent (e.g., antibody-drug conjugate, conjugate vaccine, conjugated biopharmaceutical), cell surface labeling or in vivo labeling. In some embodiments, the method of transpeptidation is a method of labeling a protein or peptide. In this method, a transpeptidase is used to link the donor substrate to the acceptor protein or peptide. The term "use of a transpeptidase to connect" as used herein is intended to mean that the donor a