Method of transpeptidation

Abstract

The present invention provides a method of transpeptidation, a transpeptidation product obtainable from the method, and a chimeric fusion polypeptide. An exemplary method of transpeptidation includes linking a donor substrate to a recipient protein or peptide using a transpeptidase, where the donor substrate is a protein, peptide, amino acid amide, or amino acid ester, and where the linking reaction produces a linking product and a by-product as a substrate for the transpeptidase; and cleaving one or more amino acids from the end of the by-product using an exopeptidase such that the truncated by-product is not a substrate for the transpeptidase, thereby moving the reaction equilibrium of the transpeptidation reaction to the ligation product.

Description

Technical Field

[0001] This invention relates to a method for transpeptide transfer. Background Technology

[0002] Site-specific labeling of proteins is a widely used tool in both industrial and academic research. Peptidases, such as sortases, butelases, and asparaginyl endopeptidases, provide efficient strategies 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. Numerous improvements to these methods have been reported, including the evolution of enzymes with enhanced and altered reactivity, the use of multiple enzymes to achieve orthogonal labeling reactions, and the development of methods for labeling internal residues. However, in almost all cases, an excess of one reaction component is required to achieve complete conversion of the unlabeled protein to the labeled protein.

[0003] Peptidases recognize specific peptide motifs in their substrate proteins or peptides. A cysteine ​​residue at the active site reacts with this motif to generate a thioacryl intermediate, which then reacts with the substrate peptide or protein to produce the product. A key challenge in such labeling reactions is that they are typically completely reversible. Figure 1 A). The enzyme's substrate recognition motif (e.g., LPXT / G for sorting enzymes) remains in the reaction product, and the byproducts generated during the initial thioacryl intermediate formation are also substrates for the second step. The reversibility of the reaction places it under thermodynamic control, resulting in an equilibrium mixture of labeled and unlabeled products. Figure 1 B). The generation of pure labeled proteins typically requires a large excess of labeled peptides and / or the removal of unlabeled proteins from the reaction mixture. For N-terminal labeling, the use of ester / depsipeptide substrates has been reported, where the byproduct alcohol is no longer a substrate of the enzyme. Figure 1 C). However, this strategy is not suitable for C-terminal labeling because it requires the incorporation of ester bonds into the expressed protein. Therefore, several alternative strategies have been developed for C-terminal labeling. For example, by using a centrifugal filtration device (5), reacting the product peptide with chemical reagents (6), or using a high concentration of Ni. 2+ Chelation (7, 8) physically removes byproduct peptides for C-terminal labeling. In other work, substrate preference of asparagine endopeptidases has been utilized to achieve selective reactions (9).

[0004] However, none of these previous strategies allow for one-pot controlled quantitative peptide ligation using transpeptidases, which shift the reaction equilibrium toward the ligation product and are applicable to both N-terminal and C-terminal labeling. Summary of the Invention

[0005] In a first aspect, a method for transpeptidation is provided, comprising: using a transpeptidase to link a donor substrate to a recipient protein or peptide, wherein the donor substrate is a protein, peptide, amino acid amide, or amino acid ester, and wherein the linking reaction produces a linking product and a byproduct that is a substrate of the transpeptidase, and then using an exopeptidase to cleave one or more amino acids from the end of the byproduct such that the truncated byproduct is not a substrate of the transpeptidase, thereby shifting the reaction equilibrium of the transpeptidation reaction toward the linking product.

[0006] The inventors have developed a coupled enzyme transpeptidase system comprising a combination of transpeptidase and exopeptidase. Advantageously, byproducts are degraded by the exopeptidase, thereby removing byproducts that serve as substrates for the transpeptidase. Therefore, the reaction equilibrium shifts to the right, facilitating product ligation and preventing reaction reversibility. This method significantly enhances the efficiency of transpeptidation reactions, promoting their wider application in experimental and industrial processes. Furthermore, the coupled enzyme transpeptidase system enables the quantitative fusion of N-terminal and C-terminal labeling of proteins or peptides with the quantitative expression of the peptide or protein.

[0007] Transpeptide transfection methods are applicable to a variety of applications, including but not limited to protein or peptide labeling, conjugation of proteins or peptides to therapeutic agents (e.g., antibody-drug conjugates, conjugated vaccines, conjugated biologics), cell surface labeling, or in vivo labeling. In some embodiments, transpeptide transfection methods are methods for labeling proteins or peptides.

[0008] In this method, a transpeptidase is used to link a donor substrate to a recipient protein or peptide. The term "linked using a transpeptidase..." as used herein is intended to mean that the donor and recipient are joined together by forming an amide bond between a free amino group and a carbonyl group under the catalysis of the transpeptidase.

[0009] The donor substrate can be any suitable protein, peptide, amino acid amide (e.g., glycine amide), or amino acid ester (e.g., glycine ester) that provides an acyl donor group for the transpeptidation reaction. In some embodiments, the donor substrate is a protein, peptide, glycine amide, or glycine ester. In some embodiments, the donor substrate is a protein, peptide, or glycine amide. In some embodiments, the donor substrate is a protein or peptide. In some embodiments, the donor substrate is a peptide. The term "protein" as used herein refers to a macromolecule comprising one or more polypeptides and includes native proteins, synthetic proteins, and modified proteins (e.g., polypeptides linked to non-peptides, such as labeled proteins, biotinylated proteins, PEGylated proteins, click-modified proteins, proteins containing non-naturally occurring amino acids, etc.). The term "peptide" as used herein refers to a molecule comprising an amino acid chain and includes native peptides, synthetic peptides, and modified peptides (e.g., peptides linked to non-peptides, such as labeled peptides, peptides containing non-naturally occurring amino acids, etc.). As used herein, the term "amino acid amide" refers to an amide of an amino acid, and includes both naturally occurring and non-naturally occurring amide forms of amino acids, which may or may not be modified. The term "amino acid ester" as used herein refers to an ester of an amino acid, and includes both naturally occurring and non-naturally occurring ester forms of amino acids, which may or may not be modified.

[0010] The receptor protein or peptide can be any suitable protein or peptide that provides an acyl receptor group for the transpeptide reaction. In some embodiments, the receptor is a receptor protein.

[0011] Transpeptidases can be any suitable enzyme that links a donor substrate to a recipient protein or peptide via a transpeptidation reaction (i.e., catalyzes the formation of an amide bond between a free amino group and a carbonyl group). Typically, a transpeptidase cleaves a recognition motif in the recipient protein or peptide to form an acyl-enzyme intermediate; the nucleophilic attack of the N-terminal amine of the donor substrate on the acyl intermediate releases the enzyme and leads to the formation of the amide bond, resulting in the linker product and byproducts.

[0012] For example, the transpeptidase can be a transpeptidase enzyme (e.g., a sorting enzyme, butelase, asparagine endopeptidase, ligase) or an enzyme engineered to have transpeptidase activity (e.g., trypsiligase, subtiligase, peptiligase, omniligase-1). In some embodiments, the transpeptidase is a cysteine ​​transpeptidase. In some embodiments, the transpeptidase is a sorting enzyme, butelase, or asparagine endopeptidase. In some embodiments, the transpeptidase is a sorting enzyme. In some embodiments, the transpeptidase is sorting enzyme A. In some embodiments, the transpeptidase is a Staphylococcus aureus sorting enzyme or an evolved functional form thereof. In some embodiments, the transpeptidase is Staphylococcus aureus sorting enzyme A or an evolved functional form thereof. Evolved sortases are known to those skilled in the art; see, for example, Podracky, CJ, An, C., DeSousa, A. et al., Laboratory evolution of a sortase enzyme that modifies amyloid-β protein. Nat Chem Biol 17, 317–325 (2021) (incorporated herein by reference in its entirety). In some embodiments, the transpeptidase is Streptococcus pyogenes sortase A.

[0013] Transpeptide reactions produce linker products and byproducts. As used herein, the term "linker product" is intended to mean a product from the transpeptide reaction containing a newly formed amide bond (i.e., a conjugated donor-acceptor product). The term "byproduct" as used herein is intended to mean a product released from the transpeptide reaction that is not a linker product. Byproducts may also be referred to as "leaving groups." In some embodiments, the byproduct is a peptide, an amino acid amide (e.g., glycine, N-ethylglycine), an amino acid ester (e.g., glycyl ester), or a peptide-like molecule. In some embodiments, the byproduct is a peptide or glycine. In some embodiments, the byproduct is a peptide.

[0014] The byproduct is a substrate of transpeptidase, meaning that transpeptidase can catalyze the reverse reaction, leading to the regeneration of donor substrate and acceptor protein or peptide. In the theoretical reverse reaction, the linker binds to transpeptidase to form an acyl-enzyme intermediate; the N-terminal amine of the byproduct nucleophilically attacks the acyl-enzyme intermediate, releasing the enzyme and causing peptide bond formation, thereby reforming the donor substrate and acceptor protein or peptide.

[0015] In this method, an exopeptidase is used to cleave one or more amino acids from the end of the byproduct. The term “cleave with an exopeptidase…” as used herein is intended to mean that the exopeptidase catalyzes the hydrolysis of the terminal or penultimate peptide bond of the byproduct to release one or more amino acids, forming a truncated byproduct.

[0016] An exopeptidase can be any suitable enzyme that cleaves one or more amino acids from the end of a byproduct to produce a truncated byproduct that is not a substrate of a transpeptidase.

[0017] In some embodiments, one or two amino acids are cleaved from the end of the byproduct. In some embodiments, one amino acid is cleaved from the end of the byproduct. In some embodiments, no more than one amino acid is cleaved from the end of the byproduct. In some embodiments, no more than two amino acids are cleaved from the end of the byproduct.

[0018] The byproduct can be N-terminus or C-terminus. In some embodiments, one or more amino acids are cleaved from the N-terminus of the byproduct.

[0019] The exopeptidase can be an aminopeptidase or a carboxypeptidase. In some embodiments, the exopeptidase is a carboxypeptidase, wherein one or more amino acids are cleaved from the C-terminus of the byproduct. In some embodiments, the exopeptidase is an aminopeptidase, wherein one or more amino acids are cleaved from the N-terminus of the byproduct.

[0020] Various aminopeptidases are known to those skilled in the art, such as D-aminopeptidase, aminopeptidase W, XXX-prolyl aminopeptidase, and XXX-prolyl dipeptidase. In some embodiments, the aminopeptidase is a D-aminopeptidase. In some embodiments, the aminopeptidase is a glycyl aminopeptidase. In some embodiments, the D-aminopeptidase is human Brucella anthropi D-aminopeptidase.

[0021] In some embodiments, the aminopeptidase is sequence-specific, optionally wherein the aminopeptidase is specific for GX2-terminated peptides, wherein X2 is a small, polar or negatively charged (i.e., acidic) amino acid, or an amino acid amide (e.g., glycine). In some embodiments, X2 is selected from alanine, glycine, serine, threonine, methionine, asparagine, glutamine, tyrosine, aspartic acid, and glutamic acid.

[0022] The truncated byproducts are not substrates for transpeptidases, meaning they cannot nucleophilically attack the acyl-enzyme intermediate. Therefore, the truncated byproducts cannot be used in the reverse reaction. As used herein, the term "truncated byproduct" refers to the final product after cleavage by an exopeptidase. For example, a byproduct with a GGX terminus at its N-terminus is cleaved by glycyl aminopeptidase to produce a GX-terminated byproduct, and then cleaved a second time by aminopeptidase to produce an X-terminated byproduct. In this case, the X-terminated byproduct is not a substrate for transpeptidases.

[0023] As a result of this method, the reaction equilibrium of the transpeptide reaction shifts towards the linker product. In other words, the truncation of the byproducts prevents the transpeptide reaction from being under thermodynamic control, favoring ammonolysis over hydrolysis. In some embodiments, the method results in at least 10-fold selectivity for ammonolysis relative to hydrolysis. In some embodiments, the method results in at least 20-fold selectivity for ammonolysis relative to hydrolysis. In some embodiments, the method results in at least 30-fold selectivity for ammonolysis relative to hydrolysis. In some embodiments, the method results in at least 40-fold selectivity for ammonolysis relative to hydrolysis. In some embodiments, the method results in at least 50-fold selectivity for ammonolysis relative to hydrolysis. In some embodiments, the method results in at least 60-fold selectivity for ammonolysis relative to hydrolysis. In some embodiments, the method results in at least 70-fold selectivity for ammonolysis relative to hydrolysis. In some embodiments, the method results in at least 80-fold selectivity for ammonolysis relative to hydrolysis. In some embodiments, the method results in at least 90-fold selectivity for ammonolysis relative to hydrolysis. In some embodiments, the method results in at least 100-fold selectivity for ammonolysis relative to hydrolysis. In some embodiments, the method is carried out at near equimolar concentrations of donor substrate and acceptor peptide or protein.

[0024] In some embodiments, the N-terminus of the donor substrate contains a motif that is not an exopeptidase. In this case, the exopeptidase does not cleave one or more amino acids from the N-terminus of the donor substrate. In some embodiments, the N-terminus of the donor substrate contains the motif XX1, where X1 is a hydrophobic or positively charged amino acid. In some embodiments, X1 is selected from cysteine, phenylalanine, histidine, isoleucine, lysine, leucine, proline, arginine, valine, or tryptophan. In some embodiments, X1 is selected from phenylalanine, histidine, isoleucine, lysine, leucine, proline, arginine, valine, or tryptophan. In some embodiments, the N-terminus of the donor substrate contains the motif GX1, where X1 is a hydrophobic or positively charged amino acid; optionally, the N-terminus of the donor substrate contains the motif GV.

[0025] As used herein, the term “N-terminal…containing motif” is intended to mean that the substrate ends with this motif at the amino terminus. For example, in the case of the N-terminal containing motif GX1, the donor substrate has a glycine residue at the N-terminus, followed by an X1 residue, optionally followed by another sequence.

[0026] Typically, receptor proteins or peptides contain recognition motifs that are cleaved by transpeptidases to produce byproducts. A variety of transpeptidases and their recognition motifs are known to those skilled in the art; see, for example, Jacobitz AW, Kattke MD, Wereszczynski J, Clubb RT. Sortase Transpeptidases: Structural Biology and Catalytic Mechanism. Adv Protein Chem Struct Biol. 2017;109:223-264; MorganHE, Turnbull WB, Webb ME. Challenges in the use of sortase and other peptideligases for site-specific protein modification. Chem Soc Rev. 2022 May 23;51(10):4121-4145, which are incorporated herein by reference in their entirety. Some examples of transpeptidases include, but are not limited to:

[0027]

[0028] In some embodiments, the recognizing motif comprises LPXT / G or LPXT / GX2, such that the byproduct comprises glycine or GX2. In some embodiments, the recognizing motif comprises LPET / G or LPET / GX2. In some embodiments, X2 is a small, polar, or negatively charged amino acid. In some embodiments, X2 is selected from alanine, glycine, serine, threonine, methionine, asparagine, glutamine, aspartic acid, and glutamic acid. In some embodiments, X2 is alanine.

[0029] In some embodiments, the N-terminus of the donor substrate contains a substrate motif that is not an exopeptidase, and the recipient protein or peptide contains a recognition motif that is cleaved by a transpeptidase to produce a byproduct.

[0030] In some embodiments, the donor substrate comprises a protein having an N-terminus that is not a substrate of an exopeptidase but a substrate of a transpeptidase. In some embodiments, the donor substrate consists of a protein having an N-terminus that is not a substrate of an exopeptidase but a substrate of a transpeptidase.

[0031] In some implementations, the donor substrate comprises a protein having an N-terminal glycine residue that is not an exopeptidase.

[0032] In some embodiments, the N-terminus of the donor substrate comprises a motif GX1, wherein X1 is a hydrophobic or positively charged amino acid, and wherein: (i) the receptor protein or peptide comprises a motif X / GX2, wherein / is a cleavage site of a transpeptidase, and X2 is a small, polar, or negatively charged amino acid; or (ii) the receptor protein or peptide comprises a motif X / G-amide, wherein the amide group is -CONH2, -CONR2, or -CONHR. R represents an organic group or hydrogen.

[0033] In some embodiments, the N-terminus of the donor substrate includes the motif GX1, wherein X1 is a hydrophobic or positively charged amino acid, and wherein the acceptor protein or peptide includes the motif LPXT / GX2, wherein / is a cleavage site of a transpeptidase, and X2 is a small, polar or negatively charged amino acid or amide group.

[0034] Positively charged or hydrophobic amino acids include C, F, H, I, K, L, P, R, V, W, and Y. Small, polar, or negatively charged amino acids include A, D, E, G, M, N, Q, S, and T.

[0035] In some embodiments, the transpeptidase and exopeptidase are provided by a chimeric fusion polypeptide. For example, the chimeric fusion polypeptide may contain transpeptidase and exopeptidase domains in tandem. In some embodiments, the chimeric fusion polypeptide also contains a purification tag. As an example, the chimeric fusion polypeptide may also contain a His tag and / or a chitin-binding domain.

[0036] In some implementations, the N-terminus and / or C-terminus of the receptor protein or peptide is attached to the donor substrate. For example, a first donor substrate may be attached to the N-terminus of the receptor protein or peptide, and a second donor may be attached to the C-terminus of the receptor protein or peptide. Those skilled in the art will recognize that any suitable donor can be used, and the donor substrates may be the same or different.

[0037] In some embodiments, the byproduct and the donor substrate have the same N-terminal residue. In some embodiments, the byproduct and the donor substrate have a glycine N-terminal residue. In some embodiments, the byproduct and the donor substrate have the same N-terminal residue and different residues at a second position.

[0038] In the second aspect, transpeptide products obtainable from the methods described herein are provided. The transpeptide products may be directly obtainable from the methods described herein. In some embodiments, the transpeptide products are further modified by subsequent chemical or enzymatic reactions.

[0039] The above description of transpeptide methods also applies to this area. Transpeptide products can also be referred to as linker products.

[0040] In the third aspect, a chimeric fusion polypeptide is provided, comprising a transpeptidase domain and an exopeptidase domain. The above description of the method, and particularly of the chimeric fusion polypeptide, also applies to this aspect.

[0041] In some embodiments, the transpeptidase domain includes a sorting enzyme A domain. In some embodiments, the exopeptidase domain includes a D-aminopeptidase domain. In some embodiments, the transpeptidase domain includes a sorting enzyme A domain, and the exopeptidase domain includes a D-aminopeptidase domain.

[0042] In the fourth aspect, a method for transpeptidation is provided, comprising: using a sorting enzyme to link a donor protein or peptide to a recipient protein or peptide, wherein the donor protein or peptide contains GX1 at its N-terminus, wherein X1 is a hydrophobic or positively charged amino acid, and wherein the recipient protein or peptide contains an LPXTGX2 motif, wherein X2 is a small, polar or negatively charged amino acid, and wherein the linking reaction produces a linking product containing the LPXTGX1 motif and a byproduct containing GX2 at its N-terminus, wherein a D-aminopeptidase cleaves one or more glycine residues from the N-terminus of the byproduct such that the truncated byproduct is not a substrate for the transpeptidase, thereby shifting the reaction equilibrium of the transpeptidation reaction toward the linking product.

[0043] The above description of the method according to the first aspect also applies to this aspect.

[0044] Where applicable or not expressly waived, it is anticipated that any of the embodiments described herein can be combined with one or more other embodiments, even if such embodiments are described under different aspects of this disclosure.

[0045] The foregoing and other objects, features and advantages of this disclosure will become more apparent from the following detailed description taken with reference to the accompanying drawings. Attached Figure Description

[0046] The invention will now be described in detail by way of example only, with reference to the accompanying drawings.

[0047] Figure 1Peptide and protein labeling reactions catalyzed by sorting enzymes. A. General transpeptide reaction protocol. Sequence-specific recognition. Catalytic attack of peptide change by cysteine ​​residues produces a thioacyl intermediate, which can be cleaved by ammonolysis (producing a new peptide) or hydrolysis. B. A sorting enzyme (SaSrtA) catalyzes the transpeptidation of LPETGX in a balanced manner to recognize peptides and proteins with an N-terminal glycine motif. C. A strategy to disrupt the equilibrium of the N-terminal labeling reaction by using phenolic peptide (ester) substrates. D. The strategy investigated in this study to disrupt these equilibrations and produce quantitative labels. The addition of D-aminopeptidase leads to the sequence-specific removal of the N-terminal glycine, enabling the use of both a sorting enzyme with broad substrate specificity and an unmodified sorting enzyme controlled by the identity of the amino acid residues following the N-terminal glycine.

[0048] Figure 2 Screening reaction conditions for quantitative labeling. A. Initial reaction protocol for screening the combined activity of SpSrtA and DAP. B. Reaction protocol for screening the combined activity of 6SrtA / 7M and DAP using peptide substrates. C. Transpeptidation of model peptides via SrtA in the presence (black) and absence (gray) of D-aminopeptidase, as analyzed by HPLC. K = Dansyl lysine. In the presence of DAP, complete conversion to the product peptide was observed.

[0049] Figure 3 Screening reaction conditions for quantitative labeling. A. Example of reaction MS analysis where the nucleophilic peptide substrate is not a DAP substrate (e.g., GV-6-terminated). In the presence of DAP (black), the reaction equilibrium shifts from the peak corresponding to the unlabeled PanZ to the labeled substance, with a slight increase in the hydrolyzed substance. Labeled... The other peak is the mass adduct of PanZ; see details. Figure 7B. Screening peptide substrates in the presence and absence of DAP can identify suitable reaction pairs. 200 μM PanZ-LPETGAH6 was incubated with 400 μM of a model labeled peptide with the sequence GXSKYG in the presence and absence of 20 μM DAP, and in the presence of 10 μM Srt7M. Green = labeled, blue = unlabeled, red = hydrolyzed. C. Time dependence of C-terminal labeling of pentamer CTB using 2 equivalents of labeled peptide. Green = 1 D. Peptide concentration dependence after 2 hours of C-terminal labeling of PanZ (200 μM) and CTB (200 μM) using 50 μM Srt7M, 20 μM DAP, and a specified proportion of labeled peptide. After 4 hours, the proportion of hydrolyzed peptides significantly increased (see...). Figure 8 Green = labeled, blue = unlabeled, red = hydrolyzed. E. MS analysis of C-terminal labeling of CTB using 1.5 and 2 equivalents of the labeled peptides shown in D.

[0050] Figure 4 Optimization of N-terminal labeling conditions for proteins and comparison with the use of phenolic peptide substrates at 25°C. A. ESMS analysis of time-dependent labeling of 100 μM GVG-CTB with Srt7M (2 μM) and 1.2 equivalents (FITC) of peptide in the presence (black) and absence (gray) of DAP (10 μM). B. Comparison of N-terminal labeling of GVG-CTB with Srt7M and fluorescent peptide for 2 hours in the presence (black) and absence (gray) of DAP. C. Time-dependent labeling of 100 μM GVG-MBP with Srt7M (5 μM) and 3 equivalents of peptide substrate in the presence (black) and absence (gray) of DAP (10 μM), or in the presence of phenolic peptide substrate (blue). D. MS analysis of MBP labeled with 3 equivalents of labeling reagent for 2 hours showed almost complete conversion to the labeled product.

[0051] Figure 5Protein labeling was optimized using the chimeric SrtA-D-aminopeptidase constructs SrtH7D and SrtCH7D at 25°C and 4°C, and the enhanced labeling was demonstrated using SrtLPXSG / DAP. A. Time course for C-terminal labeling of 100 μM CTB with 25 μM SrtH7D and 200 μM peptide at 25°C. B. MS analysis after 4 hours of labeling 100 μM CTB with 25 μM SrtCH7D. C. Peptide substrate concentration dependence after 4 hours of labeling with the 100 μM MBP-LPETGA construct at 25°C. (Green = labeled, blue = unlabeled, red = hydrolyzed). D. Applying SrtH7D to the N-terminus of GVG-MBP (black) is equivalent to using a phenolic peptide substrate (blue). E. MS analysis of I27 sequence SrtH7D-dependent C-terminal labeling, TEV cleavage, and SrtLPXSG / phenolic peptide-dependent labeling. i. MS of the initial construct; ii. MS analysis of the product with C-terminal modification using SrtH7D and peptide GVSKYG; iii. MS analysis of TEV cleavage; iv. MS analysis of the product with N-terminal modification using SrtLPXSG and phenolic peptide GABA-YLPESoGG. F. Time process of N-terminal labeling of 100 μM CTB with 105 μM labeled peptide and 2 μM SrtH7D at 4 °C. Inset: MS analysis after 10 hours of labeling. G. Labeling of 100 μM GVG-CTB with 5 μM SrtLPXSG at 4 °C, with (black) and without (gray) DAP. H. SDS-PAGE analysis of the protein fusion between affimer (nominal 50 μM) with C-terminal LPETGA motif and GVG-MBP (50 μM) at 4 °C using SrtH7D (5 μM).

[0052] Figure 6 A. Initial test reaction for determining Srt7M / DAP-dependent ligation to PanZ. B. Reaction protocol. C. Exemplary data (from) labeling with 200 μM PanZ after overnight incubation of the test peptide with Srt7M at 37°C in the presence (black) and absence of DAP, followed by incubation (from) DAP. Figure 3A. Replication. In the presence of Srt 7M, the peak corresponding to PanZ-LPETGA (approximately 16200) shifts to the lower mass (approximately 15000) corresponding to the labeled protein. In the presence of dAP, a small amount of hydrolysate (approximately 15000 Da) was observed. The additional peak at approximately +100 Da is due to contamination of the protein used for initial screening by an unknown covalent adduct. C. Exemplary data for labeling 200 μM PanZ with a peptide (GASKYG), which is also a substrate of DAP. In the presence of DAP, only hydrolysis was observed.

[0053] Figure 7 A screening reaction catalyzed by Srt7M (5 μM) was performed after 200 μM PanZ-LPETGA and 400 μM peptides with the sequence GXSKYG were incubated overnight at 25°C (approximately 16 hours). The reaction was carried out in the presence and absence of D-aminopeptidase (10 μM).

[0054] Figure 8 pH-dependent C-terminal labeling of 200 μM PanZ-LPETGA and 400 μM peptide GVSKYG at 25 °C for 4 hours catalyzed by 50 μM Srt7M in the presence of 20 μM D-aminopeptidase was determined. Maximum labeling was obtained only at pH above 8.0.

[0055] Figure 9 Labeling dependence on peptide ratio. Labeling of 200 μM PanZ-LPETGA after 4 hours of catalysis by 50 μM Srt7M, 20 μM D-aminopeptidase, and a specified ratio of GVSKYG peptide at 25 °C.

[0056] Figure 10 Increasing the DAP concentration had no effect. The results were obtained after labeling 200 μM PanZ-LPETGA with 50 μM Srt7M and 400 μM GVSKYG for 2 hours at 37°C and pH 8.0 with specified proportions of D-aminopeptidase (20 μM or 80 μM final concentration).

[0057] Figure 11 Temperature and concentration-dependent studies of Srt7M labeling were conducted at 25°C and 37°C. Only minor differences in product distribution were observed with variations in temperature and catalyst concentration. (All reactions were performed using 200 μM PanZ-LPETGA and 20 μM D-aminopeptidase with specified relative proportions of Srt7M and GVSKYG peptide substrates).

[0058] Figure 12Comparative turnover rates of PanZ labeling reactions in the presence of 20% (w / v) cosolvent. All reactions consisted of 200 μM protein substrate and 400 μM labeled peptide, 50 μM Srt7M, and 20 μM D-aminopeptidase. DMSO: dimethyl sulfoxide, GLY: glycerol, PG: propylene glycol, DMA: dimethylacetamide. DMF: dimethylformamide. Red - hydrolyzed, green - labeled, blue - unlabeled.

[0059] Figure 13 Four hours after C-terminal labeling of CTB-LPETGA, the starting material was almost completely consumed and hydrolysis increased. All reactions consisted of 200 μM CTB-LPETGA, 30 μM Srt7M, and 20 μM D-aminopeptidase (red - hydrolyzed, green - labeled, blue - unlabeled) in the presence of specified molar equivalents of GVSKYG peptide.

[0060] Figure 14 A. Comparative time series labeling of 100 μM GVG-CTB using the following methods: 2 μM Srt7M and 120 μM of phenylalanine peptides with the sequence AYLEPToGG (blue); or 2 μM Srt7M, 10 μM DAP, and 120 μM of peptides with the sequence AYLEPTGG (black). B. Comparative labeling of 100 μM GVG-CTB using 1.05, 1.2, and 2 equivalents of peptide and phenylalanine peptide substrates and 2 μM Srt7M. (Red: peptide without DAP, Black: peptide with 10 μM DAP, Blue: phenylalanine peptide substrate).

[0061] Figure 15 The time process of labeling 100 μM GVG-CTB with 2 μM SrtA. AYLPETGG peptides were analyzed by ESMS at 10 μM DAP and at concentrations of 1.2 (blue), 1.5 (brown), and 2 equivalents (green).

[0062] Figure 16 Comparative C-terminal labeling of 200 μM PanZ-LPETGA was performed at 25 °C and pH 8.0 using 25 μM Srt7M + 10 μM DAP (normal), 25 μM SrtH7D, 25 μM SrtCH7D, and 200 μM labeled peptides. Red - hydrolyzed. Green - labeled, blue - unlabeled.

[0063] Figure 17The result after 4 hours of C-terminal labeling of 100 μM MBP-LPETGA-H6 with 25 μM SrtH7D and the specified ratio of labeled peptide GVSEYG.

[0064] Figure 18 The result after 4 hours of C-terminal labeling of 100 μM MBP-LPETGA-H6 with 25 μM SrtH7D and the specified ratio of labeled peptide GVSEYG.

[0065] Figure 19 At 25°C, using a specified concentration of peptide AYLPETGG-CO2H, 2 μM SrtH7D (twill), 2 μM Srt7M + 2 μM DAP (Black) and 2 μM Srt7M (gray) peptide substrate concentration dependence after N-terminal labeling of 95 μM GVG-CTB for 2 hours. Samples of 1.7 and 2.28 equivalents were tested using 10 μM DAP. All data points represent a single experiment, except where error bars are shown.

[0066] Figure 20 Concentration dependence of peptide and catalyst for C-terminal labeling of I27 using SrtH7D. A. Study of SrtH7D and peptide substrate concentrations after 3 hours of labeling with 100 μM I27-LPETGA. B. Direct comparison of labeling 100 μM I27-LPETGA with 10 μM SrtH7D and 100–200 μM peptide GVSKYG after 3 hours. Error bars represent three independent measurements.

[0067] Figure 21 The time process of labeling 100 μM GVG-CTB with 105 μM labeled peptide AYLPETGG-CO2H and 5 μM SrtH7D at pH 8 and at specified temperature and time.

[0068] Figure 22 The time dependence of C-terminal labeling of 100 μM GVG-MBP with 25 μM SrtH7D and 200 μM labeled peptide GVSEYG at 4 °C.

[0069] Figure 23 At 4 °C, with and without 10 μM DAP, the N-terminal peptide concentration (1.1 to 3 equivalents) and time dependence (24 hours to 7 days) of N-terminal labeling of 100 μM GVG-CTB with 10 μM SrtLPXSG and the labeled peptide YALPESGAG.

[0070] Figure 24 Protein fusion between Aff-LPETGA and GVG-MBP was catalyzed by A. 1% SrtH7D and B. 2% SrtH7D at 4°C.

[0071] Figure 25 SrtH7D catalyzes the protein fusion of GVG-MBP and MBP-LPETGA to form an MBP dimer. The reaction mixture consisted of 50 μM MBP-LPETGA, 75 μM GVGK-MBP, and 12.5 μM SrtH7D at pH 8 and 4 °C.

[0072] Figure 26 Characterization data of MBP-DAP. Top left: SDS-PAGE analysis of purified protein by amylose affinity chromatography; Top right: Chromatogram of purified protein by size exclusion chromatography; Bottom: Unconvolution mass spectra of purified protein analyzed by ESMS.

[0073] Figure 27 Characterization data for Srt7M. Top left: Purification of Srt7M by Ni-NTA affinity chromatography. Top right: Purification of SrtH7D by size exclusion chromatography (Superdex 75 26 / 60 column, eluted isocratically with 50 mM Tris and 100 mM NaCl at pH 8.0). Bottom: ESMS characterization of the purified SrtH7D confirms its identity.

[0074] Figure 28 Characterization data of SrtH7D. Top left: Purification of SrtH7D by Ni-NTA affinity chromatography. Top right: Purification of SrtH7D by size exclusion chromatography (Superdex 200 16 / 60 column, eluted isocratically with 50 mM Tris and 100 mM NaCl at pH 8.0). Bottom: ESMS characterization of the purified SrtH7D confirms its identity.

[0075] Figure 29 Top left: Purification of SrtCH7D by Ni-NTA affinity chromatography; Top right: Purification of SrtCH7D by size exclusion chromatography (Superdex 200 16 / 60 column, isocratic elution with 50 mM Tris and 100 mM NaCl at pH 8.0). Pure SrtCH7D was obtained from fractions eluted between 65 and 75 mL. Bottom: ESMS characterization of the purified SrtCH7D confirmed its identity.

[0076] Figure 30ESMS analysis of the purified PanZ-LPETGA construct showed the expected quality.

[0077] Figure 31 Purification of CTB-LPETGA-H6 by unfolding via Ni-NTA affinity chromatography.

[0078] Figure 32 Characterization of refolded CTB-LPETGA. Top left: SDS-PAGE analysis of CTB-LPETGA before and after size exclusion chromatography. The unboiled CTB-LPETGA sample retains its pentamer structure on the SDS-PAGE gel, confirming polymerization. Top right: Size exclusion chromatography purification of CTB-LPETGA (Superdex 75 26 / 60 column). Bottom: ESMS characterization of purified CTB-LPETGA confirms its identity.

[0079] Figure 33 Top left: Purification of MBP-LPETGA by Ni-NTA affinity chromatography. Top right: Purification of MBP-LPETGA by size exclusion chromatography (Superdex 75 26 / 60). Bottom: ESMS characterization of the purified MBP-LPETGA confirms its identity.

[0080] Figure 34 Characterization data for GVG-MBP purification. Top left: SDS-PAGE analysis of protein purified by amylose affinity chromatography. Top right: Size exclusion chromatography (Superdex 75 26 / 60) chromatogram of the protein. Bottom: ESMS analysis of the protein confirms the identity of GVG-MBP.

[0081] Figure 35 Characterization data of GVG-CTB. Top left: SDS-PAGE analysis of protein purified by Ni-NTA affinity chromatography. Top right: Size exclusion chromatography (Superdex 75 16 / 60) chromatogram of the protein. Bottom: ESMS analysis of the protein confirms the identity of GVG-CTB.

[0082] Figure 36 ESMS characterization data of Affimer-LPETGA before and after iodoacetamide capping.

[0083] Figure 37Characterization data for (TEV)-GVG-I27-LPETGA-H6. Top left: SDS-PAGE analysis of the protein after Ni-NTA affinity chromatography purification. Top right: Size exclusion chromatography (Superdex 75 26 / 60) chromatogram of the protein. Bottom: ESMS analysis of the protein confirms the identity of (TEV)-GVG-I27-LPETGA-H6.

[0084] Figure 38 Example analysis of HPLC data.

[0085] Figure 39 Unoptimized N-terminal labeling was performed using SrtH7D at low protein substrate concentrations. The reaction mixture consisted of 10 mM GVG-CTB incubated with a specified concentration of the substrate peptide AYLPETGG at pH 8 and 4°C.

[0086] Figure 40 Unoptimized C-terminal labeling was performed using SrtH7D at low protein substrate concentrations. The reaction mixture consisted of 10 mM CTB-H6 incubated with specified concentrations of substrate peptide GVSKYG and SrtH7D at pH 8 and 4 °C for 4 h and 24 h. Labeling degree was analyzed by SDS-PAGE and ESMS. Green: labeled product, blue: unlabeled product, red: hydrolysis product.

[0087] Figure 41 Percentage N-terminal labeling of protein constructs determined by ES-MS. All experiments corresponded to incubation of 100 mM substrate protein with 2 mM SrtH7D and 150 mM labeled peptide AYLPETGG at 4°C for 24 h.

[0088] Figure 42 Exemplary data on the kinetic characterization of SrtH7D using a peptide assay based on fluorescence non-quenching targeting transpeptidase activity. 50 μM Abz-GALPETGAK(DNP)-NH2, 500 nM SrtH7D, 50 mM Tris, pH 8.0 (… The substrate peptide was incubated with 100 mM NaCl at 25°C and 100 mM NaCl at a specified concentration. (Abz: 2-aminobenzoyl, DNP: 2,4-dinitrophenyl).

[0089] Figure 43 Comparison of SrtH7D with individual components in peptide-based assays. 50 μM Abz-GALPETGAK(DNP)-NH2, 500 nM catalyst (SrtH7D, Srt7M, or Srt7M+DAP), 50 mM Tris, pH 8.0 (… The substrate peptide was incubated with 100 mM NaCl at 25°C and 100 mM NaCl at a specified concentration. Srt7M - blue triangle, Srt7M+dAP - red circle, SrtH7D - black square. All data were fitted to the Michaelis-Menten equation for a single substrate.

[0090] Figure 44 The storage stability of SrtH7D at 4°C (M to P), -20°C (Q to T), and -80°C (U to X) in the presence of increased concentrations of glycerol (0%, 10%, 20%, and 40%, respectively) was determined by peptide-based assays. Similar activity was observed in all samples except those stored with 40% glycerol after 1 and 5 weeks of storage.

[0091] Figure 45 Comparison of SrtH7D activity after approximately 1 year and 3 years of storage at -80°C. 50 μM Abz-GALPETGAK(DNP)-NH2, 500 nM SrtH7D, and 50 mM Tris were added at pH 8.0. The substrate peptide was incubated with 100 mM NaCl at 25°C and 100 mM NaCl at a specified concentration. Four samples of SrtH7D were compared (freshly purified - blue triangles, approximately 1 year old - red circles and black squares, and approximately 3 years old - green triangles). All data were fitted to the Michaelis-Menten equation for a single substrate.

[0092] Figure 46 The catalyst, temperature, and peptide equivalence dependence of C-terminal labeling of CTB-LPETGAH6 using a combination of Srt5M and D-aminopeptidase after 24 hours (top) and 48 hours (bottom) were determined by mass spectrometry. The reaction (100 μC CTB-LPETGAH6, catalyst Srt5M, Damp (D-aminopeptidase), peptide GVSEYG, and temperature) are shown. After 48 hours, optimal labeling was observed at the lowest concentration of Srt5M and at the lowest temperature, where optimal labeling was hardly affected by the concentration of either peptide or D-aminopeptidase.

[0093] Figure 47Exemplary data on protein labeling using SrtH5D. Top: Labeling of MBP-LPETGAH6 using equimolar labeled peptides. 200 μM MBP-LPETGAH6 was incubated with 200 μM GVSEYG and 20 μM SrtH5D at 4°C for 30 h. Bottom: Initial screening data on protein labeling using peptides with increased equivalence. 100 μM MBP-LPETGAH6 was incubated with 2.5 μM catalyst and a specified proportion of labeled peptides at 4°C for 30 h or 65 h. Detailed Implementation

[0094] The inventors have developed a coupled enzyme transpeptidase system comprising a combination of transpeptidase and exopeptidase. Advantageously, byproducts are degraded by the exopeptidase, thereby removing byproducts that serve as substrates for the transpeptidase. Therefore, the reaction equilibrium shifts to the right, facilitating product conjugation and preventing reaction reversibility. This method significantly enhances the efficiency of transpeptidation reactions, promoting their wider application in experimental and industrial processes. Furthermore, the coupled enzyme transpeptidase system enables both quantitative N-terminal and C-terminal labeling of proteins or peptides.

[0095] In the following examples, the inventors demonstrated that aminopeptidase can be used in combination with sorting enzymes to move the transpeptidation reaction toward the ligation product. However, the invention is intended to be more broadly applicable to combinations of transpeptidase and exopeptidase.

[0096] Example

[0097] Example 1

[0098] Materials and methods

[0099] Protein overexpression and purification

[0100] MBP-DAP. A synthetic oligonucleotide encoding *Ochrobacterium anthropii* was obtained from Genscript and subcloned into pNIC-MBP to obtain an expression construct of an N-terminal His6-MBP-tagged protein under T7 promoter control. After transformation into *E. coli* BL21(DE3), cells were seeded in 400 mL of solution supplemented with kanamycin (50 μg / mL). -1 The medium was incubated in LB broth at 37°C until OD was reached. 600= 0.6, at which point protein expression was induced with IPTG (0.5 mM final concentration). The cell pellet was separated by centrifugation (10000 g, 10 min) and resuspended in 100 mM potassium phosphate, 0.1 mM EDTA, 5 mM β-mercaptoethanol, pH 7.0, followed by lysis by acoustic treatment. The clarified lysate was purified by automated Ni-NTA affinity / size exclusion chromatography using a BioRad NGC system. Briefly, the clarified lysate was applied to a 5 mL Qiagen Histrap FF column, washed with a buffer of 50 mM potassium phosphate, 100 mM NaCl, pH 7.0 supplemented with 50 mM imidazole, and eluted with the same buffer supplemented with 500 mM imidazole. The eluted protein fraction (10 mL) was directly applied to a Superdex 200 (26 / 60) column and eluted isocratically with 50 mM potassium phosphate and 100 mM NaCl at pH 7.0 to produce purified protein. Protein identity was determined by SDS-PAGE and ESMS (expected mass 100363 Da, measured mass 100379 Da), and quantification was performed by UV absorbance at 280 nm (εtheor = 147710 M). -1 cm -1 ).

[0101] As described by Morgan et al. (22), SrtLPXSG and SaSrtA were overexpressed and purified.

[0102] SpSrtA. The expression construct of SpSrtA (23) was obtained from Addgene (51139) and transformed into E. coli BL21 (DE3). Protein overexpression was induced by IPTG. Isolated cells were resuspended (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.5), lysed by mechanical disruption at 20 kpsi (Constant Systems cell disruptor), and the clarified lysate was purified by Ni-NTA affinity chromatography. The protein fraction was dialyzed against lysis buffer and used in its isolated state.

[0103] Srt7M. Following the previous description of SrtLPXSG (22), the synthetic gene encoding Srt7M (24) was subcloned into pET11a to obtain the C-terminal His-tagged protein. The protein was purified using the method for SrtLPXSG described by Morgan et al., but without CaCl2 in the buffer. Protein identity was determined by SDS-PAGE and ESMS (expected mass 17851 Da, measured 17851 Da), and quantified by UV absorbance at 280 nm (εtheor = 17440 M). -1 cm -1 ).

[0104] SrtH7D. The synthetic gene construct of SrtCH7 was obtained from Genscript and subcloned between the XbaI and XhoI sites of pET28a. The construct consisted of an N-terminal His tag and a chitin-binding domain located prior to the coding sequence of Srt7M, as well as an additional C-terminal MCS. The coding sequence of the chitin-binding domain was removed by sequential double digestion with EcoRI and MfeI and re-ligated to obtain SrtH7, as described by Dolan et al. (25). The coding sequence of DAP was subcloned between the NdeI and BamHI sites of the SrtH7 construct. The protein was overexpressed as previously described for Srt7M and purified using sequential or automated sequential Ni-NTA affinity chromatography / size exclusion chromatography. Protein identity was determined by SDS-PAGE, ESMS (expected mass 75923 Da, measured 75923 Da), and quantified by UV absorbance (εtheor = 94935 M). -1 cm -1 ).

[0105] SrtCH7D. The SrtCH7D expression construct was prepared according to the SrtH7D model, but the chitin-binding domain coding sequence was not removed. The protein was overexpressed and purified as for SrtH7D. Protein identity was determined by SDS-PAGE and ESMS (expected mass 81888 Da, measured 81888 Da), and quantification was performed by UV absorbance (εtheor = 116030 M). -1 cm -1 ).

[0106] PanZ-LPETGA. The expression construct of PanZ-LPETGAH6 was obtained from pBAD24-Yhhk via two-step cloning (26). First, the coding sequence of PanZ (also known as YhhK) was amplified by PCR, and then the NheI / XhoI fragment was subcloned into the XbaI and XhoI sites of pET28a to generate an expression construct of PanZ with the C-terminal SLPETGAGLEH6 additional sequence. Subsequently, site-directed mutagenesis was performed using Quikchange to generate the C-terminal sequence SLPETGALEH6. The protein was overexpressed and purified as previously described for unmodified C-terminal hist-tagged proteins. Protein identity was determined by SDS-PAGE and ESMS (expected mass 16224 Da, measured 16224 Da) and quantified by UV absorbance at 280 nm (εtheor = 26470 M). -1 cm -1 ).

[0107] CTB-LPETGA-H6. The inclusion body-based expression construct of CTB-LPETGA-H6 was generated by mutagenesis of the expression construct of soluble CTB-LPETGA (49), which is itself a derivative of pMalp5x. In short, site-directed mutagenesis was used to remove the coding sequence of the LTIIb signal peptide, insert a start methionine codon, and insert a C-terminal Ser-His6 sequence. The resulting plasmid was transformed into E. coli BL21 (DE3). The transformed cells were seeded in LB medium and incubated at 37°C until OD. 600 = 0.6, inducing protein overexpression (0.5 mM IPTG) and incubating the medium overnight at 25°C (approximately 16 hours). Cells were separated by centrifugation, resuspended in 50 mM sodium phosphate 300 mM NaCl buffer (pH 8.0), and lysed by mechanical disruption. Cell debris was separated by centrifugation, and the precipitate was resuspended in 100 mM NaH2PO4, 100 mM Tris, 8 M urea, pH 8.0 (resuspending buffer), and then centrifuged again. The clarified solution was applied to a Ni-NTA affinity column (gravity feed) and the column was filled with resuspending buffer supplemented with 25 mM imidazole and 0.1% v / v Triton-X (…). Figure 31 Wash 1) and wash with resuspension buffer supplemented with 25 mM imidazole (wash 2), then elute with resuspension buffer supplemented with 400 mM imidazole.

[0108] MBP-LPETGA-H6. The expression construct of MBP-LPETGA-H6 was created by site-directed mutagenesis of plasmid pMalc5x to encode the C-terminal SSNLPETGASH6. The sequence was obtained by inserting oligonucleotides into the adapter region of the pMalc5x plasmid. The protein was overexpressed in *E. coli* BL21 using a standard protocol for IPTG-induced overexpression. Isolated cells were resuspended in 50 mM Tris, 150 mM NaCl, pH 8.0, mechanically lysed, and the clarified lysate was purified by affinity chromatography with Ni-NTA (eluted with 250 mM imidazole) or amylose (eluted with 100 mM glucose), followed by further purification by size exclusion chromatography (Superdex 200 26 / 60, moderately eluted in 50 mM Tris, 150 mM NaCl, pH 8.0). Protein identity was determined by SDS-PAGE and ESMS (expected mass 42336 Da, measured 42333 Da), and quantified by UV absorbance at 280 nm (εtheor = 66350 M). -1 cm -1 ).

[0109] GVG-MBP. The GVG-MBP expression construct was constructed by mutagenesis of pMalc5x to insert a nucleotide encoding the GVGK tetrapeptide after the start methionine codon and a stop codon before the region encoding the polyasparagine linker. After transformation into E. coli BL21, cells were cultured at 37°C and protein overexpression was induced at OD600 with IPTG (1 mM), followed by further growth for 3 to 4 hours. Cells were separated by centrifugation, resuspended in 50 mM Tris, 150 mM NaCl (pH 7.6), and mechanically lysed (using a constant systems cell disruptor). The clarified lysate was purified by amylose affinity chromatography (eluting with 10 mM maltose) and size exclusion chromatography, identified by SDS-PAGE and HRMS (expected mass 40449 Da, measured 40448 Da), and protein concentration was estimated using UV absorbance (εtheor = 66350 M). -1 cm -1 ).

[0110] GVG-CTB. The GVG-CTB overexpression construct was generated by site-directed mutagenesis of an existing GVG-CTB overexpression construct, via the LTBII leader sequence in the pMal backbone for periplasmic export. After transformation into *E. coli* BL21, cells were cultured in LB medium and protein overexpression was induced with IPTG. The medium was clarified by centrifugation (10,000 g, 10 min), and the protein was separated by precipitation with ammonium sulfate (570 g / L) and centrifugation (10,000 g, 25 min). After resuspending in 50 mM Tris, 150 mM NaCl (pH 7.6), the protein was purified by sequential Ni-NTA affinity chromatography (utilizing the natural affinity of CTB for the resin) and size exclusion chromatography on a Superdex 75 (50 mM HEPES, 100 mM NaCl, pH 8.0). Protein identity was determined by SDS-PAGE and ESMS (expected mass 11856 Da, measured mass 11856 Da), and quantification was performed by UV absorbance at 280 nm (εtheor = 11585 M). -1 cm -1 ).

[0111] (TEV)-GVG-I27-LPETGA-H6. An expression construct containing the titin I27 domain with an N-terminal TEV cleavage sequence and a C-terminal LPETGA motif was obtained from Genscript and subcloned into pET28a. The protein was overexpressed from *E. coli* BL21(DE3) and purified by sequential Ni-NTA affinity chromatography and size exclusion chromatography (Superdex 75 26 / 60). Protein identity was determined by SDS-PAGE and ESMS (expected mass 12627 Da, measured 12626 Da), and quantification was performed by UV absorbance at 280 nm (εtheor = 8605 M). -1 cm -1 ).

[0112] Chemical Experiment

[0113] Peptide and phenolic peptide synthesis. All peptides were synthesized manually or using a Liberty Blue synthesizer using standard peptide synthesis protocols. The Oxyma Pure strategy was employed, with peptide synthesis initiated on glycine-chlorotriphenylmethyl resin or Rinkamide resin. Peptide purity was assessed by HPLC and LCMS. Peptides were purified by preparative HPLC when necessary.

[0114] Peptide labeling assay. The peptide (500 μM) was combined with SrtA (50 μM) and MBP-DAP (5 μM) in 50 mM Tris.HCl pH 8.0 supplemented with 1 mM CaCl2 and incubated at 37 °C. Samples were analyzed by LC-MS – the curves shown represent the total absorbance from 190 nm to 650 nm.

[0115] Protein labeling assays. Protein and peptide concentrations were determined by UV absorbance at 280 nm. Unless otherwise specified, standard labeling reactions were performed in an Eppendorf hot mixer with 50 mM Tris, 100 mM NaCl, pH 8.0 (corrected for incubation temperature). Reaction samples were submitted for direct analysis by LC-MS or quenched with 50% MeCN:H2O and stored at 4°C before analysis.

[0116] A dual labeling protocol for I27 was employed using SrtH7D and SrtLPXSG. GVSKYG (200 μM, 2 equivalences) was mixed with TEV-GVG-I27-LPETGAH6 (100 μM) in PBS buffer (pH 8.0). H7D (25 μM, 25 mol%) was added to the reaction mixture, bringing the total volume to 500 μL. The reaction mixture was incubated at 25°C for 2.5 h and applied to a short nickel column (approximately 3 mL of nickel-NTA resin) to remove excess sorting enzyme, followed by washing with PBS buffer. The eluent and wash fraction were concentrated and washed with PBS to remove excess peptides by percolation through a rotary filter (Amicon 3 kDa MWCO). A 10095 M... -1 cm -1 The concentration of the C-terminal labeled protein was determined by the extinction coefficient. TEV cleavage was performed by mixing the C-terminal labeled I27 (TEV-GVG-I27-LPETGVSKYG) (100 μM) with his-labeled TEV protease (20 μM, 20 mol%) and shaking at room temperature for 1 hour. Excess protease was removed by nickel chromatography, and the eluent was concentrated by percolation. 8605 M -1 cm -1 The concentration of TEV-cleaved protein was determined by the extinction coefficient. GVG-I27-LPETGVSKYG (100 μM) was N-terminally labeled by adding GABA-YLPESoGG (500 μM, 5 equivalents) to PBS buffer containing 1 mM CaCl2. SrtA (LPXSG) (20 μM, 20 mol%) was added, and the reaction mixture was incubated at 37 °C for 2 h. Each step of the reaction was analyzed by HRMS.

[0117] result

[0118] Identification of transpeptidase / aminopeptidase pairs

[0119] The inventors sought to identify a second enzyme that selectively degrades specific peptide byproducts to remove them from reaction equilibrium. The initial plan was to utilize the substrate heterogeneity of a sorting enzyme A (SpSrtA) from *Streptococcus pyogenes*, which is reported to recognize both LPXT / G and LPXT / A as substrate motifs (2). The inventors hypothesized that an enzyme comprising selectively degrading or modifying peptides having an N-terminal glycine residue would facilitate protein labeling using substrate peptides having an N-terminal alanine residue. For the second enzyme, the inventors identified a D-aminopeptidase (DAP) from *Ailuropoda spp.*, as it is known to act on both peptides containing an N-terminal D-amino acid and peptides having an N-terminal glycine residue, while peptides terminated with an L-amino acid are not substrates (10-12). The cleavage products are free amino acids and truncated peptides, and the inventors have previously shown that while glycamide and peptides terminated with a single glycyl residue are substrates of the sorting enzyme, glycine is not (4).

[0120] The inventors obtained recombinant DAP as a fusion protein with an N-terminal MBP-His marker through overexpression, and initially tested the ligation using peptide substrates with recognition sequences LPETGGE and H2N-AAS, as well as a combination of DAP and SpSrtA. Figure 2A). Although they observed the formation of the desired LPETAA-containing product, the conjugation reaction was slow, and they consistently observed the substrate LPETGGE peptide undergoing hydrolysis to produce LPET at a rate similar to that of ammonolysis to form the desired labeled product. This is likely due to the lower concentration of the AAS co-substrate peptide used in this method compared to previous reports using SpSrtA (2, 13-15). While the hydrolytic activity of SpSrtA prevented effective labeling, the inventors also noted an unexpected byproduct (LPETGE) in which a single glycine residue had been removed from the initial substrate, presumably after the sorting enzyme released GGE, and DAP removed the glycine and re-linked it with the sorting enzyme-acylase intermediate. This observation suggests that DAP activity may be influenced by subsequent residues, as the partially degraded product peptide must be present in sufficient concentration to achieve the reverse reaction. Such selectivity of DAP for D-Ala-terminated peptide substrates has been previously observed, but not all amino acids have been fully characterized (10). Therefore, the inventors incubated a library of glycine-terminated peptides with the sequence GXSKYG at 25°C for 2 hours with DAP (10 mol%). Generally, the inventors observed that when the second residue was small, polar, or negatively charged (i.e., A, D, E, G, M, N, Q, S, or T), the glycine residues from the peptides were partially or completely hydrolyzed, but when the second residue was positively charged or hydrophobic (i.e., C, F, H, I, K, L, P, R, V, W, or Y), the glycine residues from those peptides were not hydrolyzed. Subsequent screening of non-natural amino acids revealed the same pattern – in this case, the inventors were able to detect hydrolysis of Gly residues in the presence of various D-amino acids (a, f, l, m, or v), linear non-natural amino acids such as valine and GABA, and leucine, but no hydrolysis was observed in the presence of ornithine, α-methylalanine, and N-methylalanine.

[0121] The inventors hypothesize that they can utilize the selectivity of DAP for the second residue in its substrate peptide to guide the linking of glycine-terminated substrate peptides. In other words, they can use peptide sequences LPETGX1 and GX2 such that while GX1 is a DAP substrate, GX2 is not, and therefore the equilibrium product will be a peptide with the sequence LPETGX2. Figure 1 D). Therefore, they investigated the ligation of available peptide substrates with the C-terminal sequence LPETGGS and the N-terminal sequence H2N-GV, but this time they used both SaSrtA and the Ca-independent SaSrtA 7M heptamutant (Srt7M) as catalysts (1,16). Figure 2 B). In this case, the addition of DAP to the reaction mixture resulted in a transition to the formation of the desired product after 3 hours. Figure 2C). This indicates that the system did indeed catalyze the formation of selective products as expected.

[0122] Instead of optimizing the reaction at high peptide substrate concentrations, the inventors immediately turned to studying the labeling of the model protein and used a available construct (17) of the PanD regulatory protein (PanZ). This protein is generated by inserting an LPETGA motif before a C-terminal His tag. In the absence of DAP, using 2 equivalents of a GV-terminated co-substrate, the inventors initially observed approximately 50% conversion to the product after overnight incubation at 25°C. Figure 3 A gray). The addition of DAP increased product formation to 80% ( Figure 3 A black), in which the remaining protein is uniformly distributed between the unlabeled substrate and the hydrolysate. Conversely, when using a peptide as a substrate for DAP (e.g., GA-terminated), the inventors observed only the unlabeled protein or the hydrolysate formed from the thioester intermediate ( Figure 6 C). Therefore, they used the labeling of PanZ to screen potential peptide substrates: by incubating the peptide and PanZ with Srt7M in the presence and absence of DAP (C). Figure 3 B and Figure 7 When the labeled peptide was a DAP substrate, the inventors observed only hydrolyzed protein and trace amounts of unlabeled substrate. However, when the labeled peptide was not a DAP substrate, the inventors observed a shift in product distribution from unlabeled substrate to labeled product, with the proportion of hydrolyzed product varying. Based on this screening, the inventors selected peptides ending in GV42, along with the LPETGA motif, as preferred substrates for their future experiments.

[0123] C-end tag optimization

[0124] The next goal is to optimize the conditions for the labeling reaction. This involves raising the pH above pH 8. Figure 8 This resulted in >95% conversion to product, likely due to the favorable pH for ammonolysis relative to hydrolysis at a pKa higher than that of the N-terminal amine, consistent with previous results regarding the optimal pH for sorting enzyme labeling (18). The proportion of desired product also increased with increasing equivalents of the peptide used for labeling, but in the case of PanZ substrates, only marginal enhancement was observed when more than two equivalents of labeled peptide were used. Figure 3 D and 9). Changes in the amount of DAP added to the reaction do not affect the overall reaction conversion (D and 9). Figure 10 Furthermore, although increasing the temperature from 25°C to 37°C allows for a reduction in the amount of catalyst, it does not significantly alter the conversion level relative to hydrolysis. Figure 11 Finally, the inventors investigated the effect of common co-solvents on the reaction ( ). Figure 12The addition of DMSO, glycerol, and propylene glycol had no effect on the reaction, while the addition of DMA and DMF inhibited the reaction but did not terminate it.

[0125] Next, the inventors investigated the C-terminal modification of the pentachotoxin B-4 subunit (CTB), which is widely used as a tool in cell biology and neuroscience (19). The inventors had previously reported CTB constructs with a C-terminal sorting enzyme recognition site; however, periplasmic expression yielded heterologous substrates in which only 80% to 90% of the protopolymer chains contained the desired extension (20). Therefore, the inventors generated a novel C-terminal His-tagged CTB construct incorporating the LPETGA labeling motif. Cytoplasmic expression of the unfolded protein formed inclusion bodies, which were subsequently dissolved in 8M urea. Affinity purification and subsequent refolding provided homogeneous material for the labeling experiments. In this case, the inventors used automated sampling to monitor the labeling reaction in real time, using 2 equivalents of the labeled peptide, and observed a maximum of 12 (>95%) labeled peptides after approximately 2 hours. Figure 3 D and Figure 13 Reducing the number of tagged peptide equivalents to only 1.5 equivalents produced essentially the same results. Figure 3 (D, 3E). In this case, the overall level of the marker was slightly higher than the observed level of PanZ, which the inventors attributed to the longer linker between the globular protein domain of the CTB protein and the LPETG recognition motif.

[0126] Optimization of N-terminal tagging

[0127] The effectiveness of C-terminal labeling using this coupling enzyme method has been demonstrated. The inventors investigated its application to N-terminal labeling and compared it with the use of phenylethyl peptide substrates. They tested the enzyme combination using protein constructs in which the GlyVal labeling motif was added to the N-terminus of the expressed protein. The inventors initially used a single equivalence of the labeling reagent in the presence (black) and absence (gray) of 10 mol% DAP. Figure 4 The labeling of pentamer GVG-CTB (A and 4B) and 2 mol% Srt7M was studied using mass spectrometry to track the reaction. The addition of DAP to the reaction mixture shifted the reaction equilibrium from a statistically 1:1 distribution between labeled and unlabeled products to labeled only. The inventors then compared labeling using phenolic peptides with labeling using a combination of peptide and DAP (…). Figure 14 A), and observed essentially the same performance using both methods. Changes in the concentration of the labeled reagent ( Figure 14 B and Figure 15This indicates that at 25°C, only 1.2 equivalents of peptide labeling are sufficient to achieve approximately 95% labeling of the protein. Both phenylphenolic peptides and the new coupling enzyme method can lead to near-quantitative labeling within 2 hours. However, the new method is more robust due to the stability of the peptide substrate in solution compared to phenylphenolic peptides, which undergo non-enzymatic hydrolysis over time. Labeling of the model maltose-binding protein construct GVG-MBP(3) revealed a similar pattern. Figure 4 (C and 4D), where the addition of DAP again enables near-quantitative N-terminal labeling using unactivated peptides, equivalent to the results observed using phenolic peptide substrates.

[0128] Generate bifunctional catalysts

[0129] Considering that the addition and removal of two enzymes increases the chance of product contamination, and that a single reagent is easier to handle and monitor, the inventors investigated combining the activities of two enzymes into a single chimeric polypeptide. Their N-terminal and C-terminal labeling methods used different ratios of the two enzymes, but DAP activity never appeared to be a limiting factor – therefore, they anticipated that fusing equimolar amounts of the two enzymes together would not significantly affect either class of reaction. The inventors generated a fusion protein consisting of a sorting enzyme and a DAP domain tandemly, with either a his tag (SrtH7D) or a his tag and a chitin-binding domain (SrtCH7D) affinity purification tag. Both SrtH7D and SrtCH7D were expressed at high levels, and after purification, the chimeric protein was shown to be stable for short-term storage at 4°C and for storage at -20°C and -80°C.

[0130] The inventors initially compared the activities of SrtH7D and SrtCH7D with those of individual component proteins for C-terminal labeling of PanZ, and observed successful labeling with a slight increase in hydrolysis levels. Figure 16 Therefore, the inventors tested the C-terminal labeling of CTB using SrtH7D, and in this case, observed substantially the same kinetic behavior as when using the single component. Figure 5 A). SrtCH7D is also very effective, and they were able to isolate modified CTBs with high levels of labeling using only 2 equivalents of peptide without further optimization. Figure 5 B). The inventors then used this method to C-terminate MBP, resulting in a novel C-terminally modified MBP-LPETGAH6 construct for this purpose. They again observed substantially quantitative labeling using a small excess of the labeled peptide. Figure 5 C and Figure 17 The labeling efficiency differed only slightly between 1.5 and 3 equivalents of labeled peptide.

[0131] For N-terminal labeling, the inventors initially compared the activity of SrtH7D / peptide combination with the Srt7M / phenolic peptide strategy for labeling MBP - and observed comparable results using both methods and 3 equivalents of labeling reagent. Figure 5 D and Figure 18 Next, they used GVG-CTB to study the concentration dependence of the labeling reaction, comparing the labeling reaction between the combined SrtH7D construct and the individual components at equimolar concentrations. Figure 19 In this case, the inventors observed near-quantitative labeling using 1.5 to 2 equivalents of the labeled peptide at 25°C, consistent with the results for the independent components.

[0132] Finally, the inventors derived a titin I27 domain suitable for dual N-terminal and C-terminal labeling (typically used as a model protein for mechanofolding in multi-domain constructs). This incorporates a C-terminal sorting enzyme motif and a potential N-terminal GVG sequence that can be revealed by TEV protease action. Quantitative labeling of the C-terminus of this protein over a 2-hour timeframe using 2 to 3 equivalents of the labeled peptide and as little as 10 mol% SrtH7D is feasible. Figure 5 E and Figure 20 Then, after TEV cleavage to generate a new GVG N-terminus, the protein was labeled using an orthogonal LPEsoG phenolic peptide substrate (3) and Srt4S-9 (SrtLPXSG) to generate a dual-labeled protein. Figure 5 E).

[0133] Further research on the scope of coupled enzyme labeling systems

[0134] Having demonstrated their ability to achieve high levels of N-terminal and C-terminal labeling, the inventors sought to address three additional issues. Could they further improve labeling efficiency? Could they combine different sorting enzymes with D-aminopeptidase? And could they use this method to achieve protein fusion? Protein labeling using a combination of Srt7M and D-aminopeptidase at 25°C is already rapid and nearly equimolar in terms of protein and peptide substrate. For N-terminal labeling, a small excess of peptide is sufficient for quantitative labeling; however, for C-terminal labeling, even at relatively high label-to-protein ratios, peptide hydrolysis remains a challenge. In practice, even when the sorting enzyme selectively catalyzes ammonolysis rather than hydrolysis, small amounts of hydrolysis products are still observed because the latter reaction is irreversible. This problem could be solved by conducting the reaction at sufficiently high substrate concentrations, but this is impractical for most proteins. Therefore, the inventors sought to determine whether temperature could be used to control this side reaction.

[0135] Because they observed more hydrolysis at 37°C than at 25°C, the inventors decided to investigate the reaction at 4°C, assuming that the selectivity of ammonolysis relative to hydrolysis would be higher as the temperature decreased. They used 0.02 equivalents of SrtH7D and 1.1 equivalents of peptide, incubating overnight at 4°C, 16°C, and 25°C to test the N-terminal labeling of the GVG-CTB construct (…). Figure 21 Although slower, the reaction at 4°C eventually resulted in the same labeling levels as observed at higher temperatures. After overnight incubation, the inventors observed >99% protein labeling at 4°C, whereas at higher temperatures, hydrolysis-mediated unlabeling occurred. Variations in peptide concentration revealed that only 1.05 equivalents were required to produce substantially quantitative labeling, and time-process experiments showed that labeling levels remained stable between 10 and 24 hours under these conditions. Figure 5 F), and then the peptide label begins to be hydrolyzed and removed. In practice, these concentrations are within the error range of the methods used to determine reagent concentrations, indicating that equimolar N-terminal labeling with a sorting enzyme has been achieved for the first time. They similarly re-investigated C-terminal labeling of two MBP-LPETGAs under the same conditions, but this time using 0.25 equivalents of the SrtH7D catalyst. In this case, the inventors observed quantitative labeling of the protein using 2 equivalents of the labeled peptide, with no detectable hydrolysis (F). Figure 22 However, unlike the N-terminus, it cannot reproducibly produce the same success with close to 1 molar peptide substrate.

[0136] The inventors have previously demonstrated the orthogonality between the Srt4S-9 (SrtLPXSG) variant and WTSrtA for phenolic peptide-mediated labeling (3) and used these reagents in the dual labeling methods discussed above. Therefore, they investigated whether this Ca-dependent variant sorting enzyme could be combined with DAP for quantitative peptide labeling. The inventors screened GVG-CTB labeling at 4°C with and without DAP using various peptide labeling ratios (…). Figure 5 G and Figure 23 In all cases, they observed increased labeling relative to SrtLPXSG alone, and after 24 hours of incubation, both 2-equivalent and 3-equivalent peptide labels showed labeling >95%. This suggests that the method should be readily extensible to other Ca-dependent and non-dependent sorting enzyme variants or other transpeptidases with different substrate specificities.

[0137] Finally, the inventors considered whether SrtH7D could be applied to the fusion of two globular proteins. We had previously attempted to achieve protein fusion using sorting enzyme variants; however, at 25°C, they frequently observed hydrolysis of the N-terminal protein fragment containing the LPETG motif. Therefore, the inventors investigated fusion at 4°C using a small excess of the C-terminal fragment. Using these conditions, they observed quantitative fusion of the N-terminal fragment (in this case, the Affimer protein Aff-LPETGA (21) with the model C-terminal fragment (GVG-MBP)) at 4°C for 7 days using 10 mol% (5 μM) SrtH7D. Slower fusion was observed at 1 and 2.5 μM SrtH7D. Figure 24 ). In the dimerization of MBP ( Figure 25 Similar trends of successful connections have been observed in other fusions formed by their components.

[0138] discuss

[0139] Ideally, transpeptidation involves mixing two substrates (peptides or proteins) in equimolar concentrations in the presence of a catalyst, yielding a quantitative product. In this work, the inventors have demonstrated that a combination of a sequence-specific aminopeptidase and a sequence-specific transpeptidase is an efficient strategy for achieving such a reaction. As shown above, the broad substrate tolerance of the sorting enzyme (with respect to the second residue in the nucleophilic substrate) combined with the sequence specificity of the D-aminopeptidase used enables selective transpeptidation. Given that these two peptidases can selectively and preferentially form one peptide product, a key parameter still to be optimized is the chemoselectivity of the transpeptidase for ammonolysis relative to hydrolysis.

[0140] Established transpeptide methods typically employ a large excess of labeling to ensure conversion, making hydrolysis side reactions less significant because the substrate and byproduct peptides compete for hydrolysis. The key benefit of coupled enzymatic methods is that they minimize this excess, but avoiding hydrolysis is crucial for their success. For N-terminal labels, irreversible hydrolysis of the peptide substrate is less of a concern than for C-terminal labeled proteins because the loss due to peptide labeling hydrolysis can be easily offset with a small excess of peptide; however, for C-terminal labels, any hydrolysis will lead to an irreversible decrease in overall conversion.

[0141] At the concentrations used for protein labeling, the inventors observed that ammonolysis showed approximately 100-fold selectivity relative to hydrolysis at 25°C. Given the relative concentrations of the labeled peptide (200 μM) and water (55 M), this roughly corresponds to 2.5 × 10⁻⁶ at that temperature. 6 The selectivity of ammonolysis relative to hydrolysis is several times that of the two catalytic pathways, which is approximately 40 kJ / mol. -1The energy barrier difference. Given this difference, lowering the temperature to 4°C is expected to increase the selectivity of the reaction by 3 to 4 times. This estimate is largely consistent with the small increase in selectivity observed. Further increasing this ratio would require the use of different catalysts. The inventors anticipate that other natural or evolved enzymes may exhibit such selectivity at higher temperatures and lower the K0 of the ammonolysis substrate. m This will be the key parameter to be optimized to maximize reaction conversion.

[0142] As described above, for N-terminal labeling, a small amount (2 mol%) of catalyst at 25°C and a moderate (1.5 to 2-fold) excess of peptide are sufficient to achieve near-quantitative labeling. In general, this method is largely equivalent to the inventors' previous phenolic peptide method, but with the advantage of using standard peptides that are stably stored in buffer. At 4°C, although the reaction is slower, the relative stability of the product against hydrolysis means the reaction is more efficient and requires less excess peptide. Although the longer incubation time for C-terminal labeling leads to irreversible hydrolysis, the reaction mixture shows a stable product distribution over 10 to 20 hours in the presence of a small excess peptide, providing flexibility in the protein labeling workflow. The success of protein fusion experiments is particularly noteworthy in this regard, although the inventors suspect that steric hindrance in these cases could hinder the rebinding of the catalyst to the product, thereby negatively impacting the hydrolysis reaction and allowing for the use of lower catalyst concentrations.

[0143] In this study, the inventors have demonstrated the combined application of D-aminopeptidase with two different sorting enzymes; however, they anticipate that the same strategy can be readily applied to other variant sorting enzymes or transpeptidases. For example, the inventors predict that the combination of D-aminopeptidase with OaAEP1 and substrates terminated with NGM and GL or GV will work effectively.

[0144] Example 2

[0145] The inventors further considered using this method at lower protein concentrations. Quantitative N-terminal labeling can be achieved using only 1.1 or 1.5 equivalents of peptide with 10 μM protein. Figure 39 However, for C-terminal labeling, any hydrolysis leads to an irreversible decrease in overall conversion, and an excess of peptide is required to maintain the peptide / water concentration ratio. For example, using 10 μM protein, although complete consumption of unlabeled protein was observed at all peptide concentrations, 400 μM peptide was required to completely inhibit hydrolysis—this is the peptide concentration used for labeling higher concentrations of protein. Figure 3 E and Figure 40 ).

[0146] The inventors believed that their early optimization of N-terminal protein labeling (4) relied on protein labeling with a single N-terminal glycine residue, and for example, initially produced a construct (27) for labeling the cholera toxin B subunit with an N-terminal triglycine motif. Given the observed sequence specificity of D-aminopeptidase degradation of hexapeptides and the sequence requirements of C-terminal labeled peptides, they initially anticipated that these proteins would not be suitable substrates for N-terminal labeling using a combination of sorting enzymes and D-aminopeptidase. To test this hypothesis, the inventors investigated labeling of the GGG-CTB construct using the SrtH7D construct. They used optimized conditions for labeling GVG-CTB and incubated 100 μM of GGG-CTB or GVG-CTB with 2 μM SrtH7D and 150 μM AYLPETGG labeled peptides at 4°C. In both cases, they observed substantially quantitative labeling, with no unlabeled protein detected in the case of GGG-CTB. Figure 41 They hypothesized that the successful labeling was due to steric hindrance of D-aminopeptidase binding caused by the short linker region, and therefore investigated the labeling of longer proteins—specifically, the N-terminal glycine residue displayed on the N-terminal his tag in the pET28a-encoding vector, due to the action of methionine aminopeptidase, where the N-terminal sequence is GSSH… They again incubated 100 μM of the model protein (in this case, human ATIC) for 24 hours under the same conditions. They again observed complete labeling of the protein under these conditions, and therefore repeated the labeling reaction in a combined and individual manner using separate labeling proteins (Srt7M and DAP). They observed complete labeling in the presence of both enzymes, approximately 66% labeling in the presence of Srt7M alone (expected cf 60%), and no evidence of N-terminal degradation of the protein in the presence of D-aminopeptidase. To confirm the generality of this labeling, they labeled two other available proteins expressed in the same backbone (LgtC and DsbA), observing 99% and 97% labeling, respectively, under the same conditions. This suggests that the method should be effective for labeling a wider range of proteins at the N-terminus.

[0147] The inventors further characterized the transpeptidase activity of the SrtH7D fusion protein using a fluorescence dequenching-based peptide assay. Figure 42 They used this method to further compare the performance of the individual components of the chimera compared to the combined chimera, and observed a slight decrease in maximum activity compared to the individual components. Figure 43 A systematic study of systems stored at 4°C, -20°C, and -80°C showed that the residual activity remained virtually unchanged over a 5-week period at all temperatures. Figure 44Using the same assay to compare SrtH7D samples prepared at different times and stored at -80°C for 1 to 3 years, activity decreased over this extended storage period. Figure 45 Finally, the specificity of the system for the N-terminus of the model peptide was re-evaluated using a fluorescence dequenching assay (Table 1). Although some variations in activity were observed between substrates, these spanned only about a 2-fold range of specific activity.

[0148]

[0149]

[0150] Table 1. Kinetic parameters of SrtH7D-mediated peptide linkages determined by peptide-based fluorescence dequenching assays. Similar Vmax / Km values ​​were observed for all tested substrates.

[0151] The inventors investigated the combined application of D-aminopeptidase and the Ca-dependent pentavalent mutant Srt5M (28). The combined application of the two enzymes to the C-terminal labeling of CTB-H6 was studied through multivariate phylogenetic analysis (...). Figure 46 The reaction samples were quenched with EGTA before analysis using electrospray mass spectrometry. Optimal labeling was observed at 4 °C using 1% Srt5M, with little variation in either the effect of increasing the concentration of the labeled peptide from 2 equivalents to 3 equivalents or the amount of D-aminopeptidase used. A chimeric protein similar to SrtH7D (designated SrtH5D or H5D) was constructed in a similar manner, consisting of Srt5M linked to DAP. Assays of this protein showed similar performance to SrtH7D, with good labeling of MBP-LPETGAH6 observed relative to Srt5M alone. Figure 47 Given the many possible embodiments to which the principles of the disclosed invention can be applied, it should be recognized that the illustrated embodiments are merely preferred embodiments of the invention and should not be considered as limiting the scope of the invention. Rather, the scope of the invention is defined by the appended claims.

[0152] References

[0153]

Claims

1. Methods for transpeptide transfer, including: A transpeptidase is used to link a donor substrate to a recipient protein or peptide, wherein the donor substrate is a protein, peptide, amino acid amide, or amino acid ester, and wherein the linking reaction produces a linker product and a byproduct that is a substrate of the transpeptidase. Using an exopeptidase to cleave one or more amino acids from the end of the byproduct, such that the truncated byproduct is not a substrate of the transpeptidase, thereby shifting the reaction equilibrium of the transpeptidation reaction toward the linker product.

2. The method of claim 1, wherein the exopeptidase is an aminopeptidase, and wherein one or more amino acids are cleaved from the N-terminus of the byproduct.

3. The method according to claim 2, wherein the aminopeptidase is a D-aminopeptidase.

4. The method according to claim 2 or claim 3, wherein the aminopeptidase is glycylaminopeptidase.

5. The method according to claim 3, wherein the D-aminopeptidase is human Brucella anthropi D-aminopeptidase.

6. The method according to any one of claims 2 to 5, wherein the aminopeptidase is sequence-specific, optionally wherein the aminopeptidase is specific for peptides terminated at GX2, wherein X2 is a small, polar or negatively charged amino acid, or other glycine derivative.

7. The method according to any preceding claim, wherein the N-terminus of the donor substrate comprises a motif that is not a substrate of the exopeptidase.

8. The method of claim 7, wherein the N-terminus of the donor substrate comprises a motif XX1, wherein X1 is a hydrophobic or positively charged amino acid.

9. The method of claim 8, wherein the N-terminus of the donor substrate comprises the motif GX1, wherein X1 is a hydrophobic or positively charged amino acid, optionally wherein the N-terminus of the donor substrate comprises the motif GV.

10. The method according to any preceding claim, wherein the receptor protein or peptide comprises a recognition motif, the recognition motif being cleaved by the transpeptidase to produce the byproduct.

11. The method of claim 10, wherein the recognition motif comprises LPXT / G or LPXT / GX2, such that the byproduct comprises glycine or GX2, optionally wherein the recognition motif comprises LPET / G or LPET / GX2.

12. The method of claim 11, wherein X2 is a small, polar or negatively charged amino acid, optionally wherein X2 is A.

13. The method according to any preceding claim, wherein the N-terminus of the donor substrate comprises a motif that is not a substrate of the exopeptidase, and wherein the receptor protein or peptide comprises a recognition motif that is cleaved by the transpeptidase to produce the byproduct.

14. The method of claim 13, wherein the N-terminus of the donor substrate comprises the motif GX1, wherein X1 is a hydrophobic or positively charged amino acid, and wherein: (i) The receptor protein or peptide comprises the motif X / GX2, where / is the cleavage site of the transpeptidase, and X2 is a small, polar, or negatively charged amino acid; or (ii) The receptor protein or peptide contains a motif X / G-amide, wherein the amide group is -CONH2, -CONR2 or -CONHR.

15. The method of claim 14, wherein the N-terminus of the donor substrate comprises a motif GX1, wherein X1 is a hydrophobic or positively charged amino acid, and wherein the receptor protein or peptide comprises a motif LPXT / GX2, wherein / is a cleavage site of the transpeptidase, and X2 is a small, polar or negatively charged amino acid or amide group.

16. The method according to any of the preceding claims, wherein the transpeptidase is a cysteine ​​transpeptidase.

17. The method according to any of the preceding claims, wherein the transpeptidase is a sorting enzyme, butelase, or asparagine endopeptidase.

18. The method of claim 17, wherein the transpeptidase is a sorting enzyme.

19. The method of claim 18, wherein the transpeptidase is sorting enzyme A, optionally wherein the sorting enzyme A is Streptococcus pyogenes sorting enzyme A or an evolved form thereof.

20. The method according to any of the preceding claims, wherein the transpeptidase and the exopeptidase are provided by a chimeric fusion polypeptide.

21. The method according to any of the preceding claims, wherein the N-terminus and / or C-terminus of the receptor protein or peptide is linked to the donor substrate.

22. The method according to any of the preceding claims, wherein the byproduct and the donor substrate have the same N-terminal residue, optionally wherein the byproduct and the donor substrate have glycine N-terminal residues.

23. A transpeptide product that can be obtained from the method according to any one of claims 1 to 22.

24. A chimeric fusion polypeptide comprising a transpeptidase domain and an exopeptidase domain.

25. The chimeric fusion polypeptide of claim 24, wherein the transpeptidase domain comprises a sorting enzyme A domain, and wherein the exopeptidase domain comprises a D-aminopeptidase domain.

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