Cysteine protease structural domain mediated protein C-terminal selective modification method

Abstract

The invention relates to a cysteine protease structural domain mediated protein C-terminal selective modification method. The method comprises the following steps: adding a nucleophilic reagent, a catalyst and a cysteine protease allosteric activator into a target protein fused with a cysteine protease structural domain (CPD) as shown in a formula (I), and releasing a C-terminal modified target protein while inducing CPD splitting decomposition. The invention also provides a CPD-catalyzed proteolysis method and a C-terminal modification conversion method on the basis of the C-terminal modification. The method disclosed by the invention has high flexibility and has a huge application prospect in C-terminal modification of protein molecules. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of protein modification, and in particular relates to a method for selectively modifying the C-terminus of a protein mediated by a cysteine ​​protease domain (CPD). Background Art

[0002] Site-specific modification of proteins has been one of the research hotspots in the field of chemical biology in recent years, and has been widely used in drug development, in situ imaging, diagnosis, and biomaterials. Due to the complexity of protein structure, traditional chemical modification methods mainly rely on the chemical selectivity of a specific residue of the protein, which often leads to non-specific modification and makes it difficult to obtain uniform products. Therefore, it is of great significance to develop a new method for site-specific modification of proteins.

[0003] The cysteine ​​protease domain (CPD) is one of the core components of MARTX (multifunctional-auto-processing repeats-in-toxin) toxins. MARTX toxins are a large toxin family with more than 4,000 amino acid residues. They are widely present in pathogens such as Vibrio cholerae and are important tools for pathogens to resist host immune defense. After being inserted into the host cell plasma membrane, the cysteine ​​protease domain CPD can be activated by InsP6 in eukaryotic cells, undergoing conformational changes, and then cleaving the MARTX toxin through enzymatic hydrolysis and releasing effector proteins, acting on the signaling pathway of host cells. Based on this, CPD has been developed as a self-cleaving fusion tag to achieve efficient expression and purification of recombinant proteins. CPD is fused to the C-terminus of the target protein, and the obtained fusion protein can be purified by IMAC, and then InsP6 is added to trigger CPD-mediated cleavage to release the target protein. In this system, CPD can promote protein expression and solubilization on the one hand; on the other hand, it can be efficiently and specifically removed under the action of InsP6 to release the target protein, playing an important role in protein expression and purification. Summary of the invention

[0004] The purpose of the present invention is to provide a method for selectively modifying the C-terminus of a protein mediated by a cysteine ​​protease domain (CPD) and a C-terminus-modified protein obtained thereby.

[0005] In a first aspect of the present invention, a method for selectively modifying the C-terminus of a protein is provided, the method comprising the following steps 1):

[0006] A nucleophile, a catalyst and a cysteine ​​protease allosteric activator are added to the target protein of the cysteine ​​protease domain (CPD) fused with the following formula (I), and the C-terminally modified target protein is released while inducing the cleavage of the CPD:

[0007]

[0008] in

[0009] In formula (I), the cysteine ​​protease recognition sequence is an amino acid motif that can be recognized by the cysteine ​​protease domain, and its sequence is XXLXXXX, X is any amino acid, and L is leucine (Leu); preferably VDALADGK (SEQ ID No.: 1), KEALADGK (SEQ ID No.: 2), LHALGETS (SEQ ID No.: 3), LDALSGNE (SEQ ID No.: 4), KGSLSGAN (SEQ ID No.: 5), LEGLSGTN (SEQ ID No.: 6), LDWLSGKN (SEQ ID No.: 7), LQALSGKN (SEQ ID No.: 8), SGALMSQY (SEQ ID No.: 9), GAALADGK (SEQ ID No.: 10), etc.;

[0010] PRT represents the target protein to be modified, which is an amino acid sequence comprising at least 3 amino acids in length;

[0011] CPD is a cysteine ​​protease domain.

[0012] In a specific embodiment, the nucleophile is site-specifically coupled to the C-terminus of the target protein by the above method; preferably, the nucleophile is site-specifically coupled to the target protein and the CPD connection site; preferably, the nucleophile is site-specifically coupled to the C-terminus of the target protein and the CPD connection sequence valine-aspartic acid-alanine-leucine (VDAL); more preferably, the nucleophile is site-specifically coupled to the C-terminus of leucine (Leu) at the connection between the target protein and the CPD.

[0013] In a specific embodiment, the method is:

[0014] Adding a nucleophile, a catalyst and a cysteine ​​protease allosteric activator to the target protein fused with a cysteine ​​protease domain (CPD) as shown in formula (I), and after the reaction is complete, purifying to obtain the C-terminally modified target protein;

[0015] In a specific embodiment, PRT represents the target protein to be modified. In a specific example, it can be derived from mutant endoglycosidase Endo-F3 D165A, Her2 antibody, green fluorescent protein GFP, endoglycosidase Endo-A, endoglycosidase Endo-S2, glutathione S-transferase GST, and the like.

[0016] In a specific embodiment, the nucleophilic agent has a structure shown in the following formula (II):

[0017] NH2-XR(II)

[0018] in

[0019] X is absent or is a linking group selected from an unsubstituted or substituted alkylene group, a polyethylene glycol group, wherein the carbon in the alkylene backbone may be optionally replaced by S, N heteroatoms; and preferably, the substituent is selected from (=O), C1-C6 alkyl;

[0020] R is a reactive group that can be further reacted or modified, a biologically active group, or a detectably reactive group.

[0021] In a specific embodiment, R is a bioorthogonal group.

[0022] In a specific embodiment, R can be selected from the following groups: an azide group, an amino group, an alkynyl group, an amino group, a hydroxyl group, a thiol group, a sulfonic acid group, a trans-cyclooctene group, and the like.

[0023] In a specific embodiment, R is a group derived from an amino acid, a peptide chain, a protein, a sugar, a fluorescent molecule, a metal ion, a nucleotide, or the like.

[0024] In a specific embodiment, R is a group derived from a drug or a drug-linker, for example, the drug is a tumor therapeutic drug.

[0025] In a specific embodiment, R is a group derived from a fluorescent molecule.

[0026] In a specific embodiment, when a nucleophilic reagent of the above formula (II) is used, the C-terminally modified target protein obtained by the method has a structure shown in the following formula III:

[0027]

[0028] wherein the definitions of PRT, X, and R are the same as those in the preceding text, respectively; the cysteine ​​protease recognition sequence 1 is a truncated sequence of the cysteine ​​protease recognition sequence in formula (I), having the following general formula XXXL, wherein X is any amino acid, and L is leucine (Leu); specifically, the cysteine ​​protease recognition sequence 1 is VDAL (SEQ ID No.: 11), LDAL (SEQ ID No.: 12), LHAL (SEQ ID No.: 13), KGSL (SEQ ID No.: 14), LEGL (SEQ ID No.: 15), LDWL (SEQ ID No.: 16), LQAL (SEQ ID No.: 17), SGAL (SEQ ID No.: 18), GAAL (SEQ ID No.: 19) or KEAL (SEQ ID No.: 20).

[0029] In a specific embodiment, the nucleophile can be selected from any one of the following:

[0030]

[0031] In a specific embodiment, the catalyst refers to a substance that can increase the reaction rate, and can be selected from any of the following:

[0032]

[0033] In a specific embodiment, the cysteine ​​protease allosteric activator refers to a substance that can induce conformational changes in the cysteine ​​protease domain (CPD), thereby promoting CPD self-cleavage, which can be selected from inositol hexaphosphate (InsP6) and its hydrolysis products, specifically, can be selected from any of the following:

[0034]

[0035] In a specific embodiment, the method further comprises the step of isolating and purifying the released C-terminally modified target protein.

[0036] In a specific embodiment, the method further comprises a hydrolysis step 2) after step 1):

[0037] The C-terminally modified protein of formula (III) is reacted with cysteine ​​protease CPD and a cysteine ​​protease allosteric activator to obtain a hydrolysis product of formula (III).

[0038] In a specific embodiment, the hydrolyzate of formula (III) has a structure shown in the following formula (IV):

[0039] PRT-XXXL-COOH(IV)

[0040] Wherein, X is any amino acid, and L is leucine.

[0041] In a specific embodiment, the hydrolysis product of formula (III) has the following structure: PRT-VDAL-COOH.

[0042] In a specific embodiment, in the step 2), the final concentration of cysteine ​​protease in the reaction system may be 2 μM-500 μM, and the final concentration of cysteine ​​protease allosteric activator may be 2 μM-500 μM.

[0043] In a specific embodiment, in step 2), the pH of the reaction system may be 7.4-8.5, and the buffer may be selected from phosphate buffer, sodium acetate buffer, Tris-HCl buffer, PIPES buffer, MES buffer, HEPES buffer, BES buffer, etc.

[0044] In a specific embodiment, in step 2), the reaction temperature may be 4° C.-37° C., and the reaction time may be 10 min-10 h.

[0045] In a specific embodiment, the method further comprises a step 3) of converting the protein C-terminal modification after step 1):

[0046] In the presence or absence of a catalyst, the C-terminally modified protein of formula (III) is reacted with other nucleophiles, cysteine ​​protease CPD and cysteine ​​protease allosteric activator to obtain a target protein having a C-terminal modified structure different from the modified structure of step 1), wherein the other nucleophile is a nucleophile different from the nucleophile used in step 1).

[0047] In a specific embodiment, in step 3), there is no need to perform side chain protection on the protein to be modified.

[0048] In a specific embodiment, in step 3), in the reaction system, the final concentration of the nucleophile may be 2mM-100mM; the final concentration of the catalyst (if present) may be 0-10mM, the final concentration of the cysteine ​​protease may be 2μM-500μM, and the final concentration of the cysteine ​​protease allosteric activator may be 2μM-500μM.

[0049] In a specific embodiment, in the step 3), the pH of the reaction system may be 7.4-8.5, and the buffer may be selected from phosphate buffer, sodium acetate buffer, Tris-HCl buffer, PIPES buffer, MES buffer, HEPES buffer, BES buffer, etc. Preferably, the buffer may be PBS phosphate buffer, sodium acetate buffer or MES buffer.

[0050] In a specific embodiment, in step 3), the reaction temperature may be 4° C.-37° C., and the reaction time may be 10 min-10 h.

[0051] In a specific embodiment, the cysteine ​​protease allosteric activator used in steps 2) and 3), the other nucleophilic reagent and the catalyst used in step 3) are all as defined above.

[0052] It should be particularly noted that the above steps 2) and 3) are only used for numbering to distinguish the steps from each other, and do not indicate the order in which the steps are performed. In other words, depending on the C-terminal modification structure of the modified protein to be obtained, step 2) can be optionally performed after step 1), and step 3) can also be optionally performed.

[0053] In a specific embodiment, when R in the C-terminally modified target product of formula (III) is a bioorthogonal group, the method may further include a secondary modification step:

[0054] The C-terminally modified target protein of formula (III) is further reacted with a functional molecule or a pharmaceutically active molecule to obtain a product in which the C-terminus of the target protein is secondarily modified.

[0055] In another aspect, the present invention provides a C-terminally modified target protein obtained by the above method.

[0056] Beneficial Effects

[0057] In summary, the present invention provides a method for cysteine ​​protease domain (CPD)-mediated protein C-terminal diversification derivatization, including the following three modification strategies:

[0058] (I) A CPD-mediated selective modification strategy for protein C-terminus:

[0059] a. Fusing CPD to the target protein to obtain a target protein-CPD fusion protein;

[0060] b. Adding a nucleophile and a cysteine ​​protease allosteric activator to the fusion protein obtained in the previous step to induce CPD cleavage and release the C-terminal functionally derivatized target protein;

[0061] c. Isolate and purify the C-terminally modified target protein.

[0062] (II) A CPD-catalyzed proteolysis strategy:

[0063] Mixing CPD, cysteine ​​protease allosteric activator and the target protein modified at the C-terminus by strategy (I) can achieve hydrolysis of specific sequences of the target protein.

[0064] (III) A CPD-catalyzed protein C-terminal modification conversion strategy:

[0065] The CPD, the cysteine ​​protease allosteric activator and the nucleophilic reagent are mixed with the target protein with C-terminal modification obtained by strategy (I), so as to achieve the modification of the C-terminal of the target protein or the conversion of the C-terminal functional group.

[0066] The method of the present invention has high flexibility and a wide range of applications. It can be used for the coupling of protein molecules with drug molecules, fluorescent molecules, carbohydrate compounds, etc., and the preparation of nano antibody drug conjugates (NDCs) and double antibodies, for the synthesis of protein fluorescent labeling, glycoproteins, C-terminal-C-terminal protein fusions, etc., in addition, the excision of the modified functional group at the C-terminal end of the protein can be achieved, for example, the shearing of the label and the quenching of fluorescence can be achieved. Furthermore, the method can also flexibly achieve the exchange of functional molecules at the C-terminal end of the protein. Therefore, the method provided by the application has a huge application prospect in the C-terminal modification of protein molecules. DETAILED DESCRIPTION

[0067] Hereinafter, the method of the present application is described in detail through specific embodiments so that those skilled in the art can better understand the present invention.

[0068] the term:

[0069] Cysteine ​​protease domain: cysteine ​​protease domain, abbreviated as CPD

[0070] In the following prepared C-terminal modified protein structural formulas, Represents mutant endoglycosidase Endo-F3 D165A, Indicates Her2 nanoantibody, Green fluorescent protein GFP, Endoglycosidase Endo-A indicates glutathione S-transferase GST, and Indicates endoglycosidase Endo-S2.

[0071] Materials and instruments

[0072] The CPD-labeled target protein used in the present invention is expressed in the E. coli system, and the pET22b-CPD plasmid is purchased from Suzhou Jinweizhi Biotechnology Co., Ltd. The pET26b-SDAB-Her2 nanoantibody gene and the pCMV6 AC-GFP gene are provided by the Shanghai Institute of Materia Medica. The pGEX5 GST, pCW Endo A, pET26b-Endo-S2 and pET22b-CPD Endo-F3 genes are synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., all designed primers are synthesized by Sangon Biotech (Shanghai) Co., Ltd., Trans5α and BL21 (DE3) chemically active cells are purchased from Beijing Qingke Biotechnology Co., Ltd., and other reagents and materials used for expression and purification are purchased from Sangon Biotech (Shanghai) Co., Ltd. unless further explained. Sodium phytate (InsP6), propargylamine, 3-azidopropylamine, glycine, diglycine, glycine-glycine-glycine, biotin, 4-pyrrolidinopyridine, 4-mercaptophenylacetic acid, and fluorescein isothiocyanate used in the invention were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., amino acid compounds were purchased from Shanghai Jier Biochemical Co., Ltd., small molecule cytotoxic drug MMAE was purchased from Shanghai Ruishan Pharmaceutical Technology Co., Ltd., DBCO compounds and BCN compounds were purchased from Shanghai Fuqi Pharmaceutical Technology Co., Ltd., and other compounds and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. unless further explained.

[0073] The instruments and chromatographic columns used in the following text include: Waters Xevo G2-XS QTOF, liquid chromatography-mass spectrometry (LC-MS), Waters Xevo G2-XS QTOF, equipped with Waters C4 (ACQUITY UPLC Protein BEH C4, 1.7 μm, 2.1 mm x 50 mm).

[0074] Example 1: Preparation of protein C-terminal derivative P1

[0075]

[0076] The mutant endoglycosidase Endo-F3 D165A-CPD (SEQ ID No.: 22) labeled with CPD (SEQ ID No.: 21), 3-azidopropylamine, 4-dimethylaminopyridine (DMAP) and phosphoinositide (InsP6) were dissolved in 50mM sodium acetate solution (pH = 7.4) to make the concentrations of each component 100μM, 5mM, 5mM, and 5μM, respectively. After incubation at 4°C for 3h, Ni-NTA agarose resin was added and incubated at 4°C for 2h. The His-tagged CPD was bound, filtered, and the filtrate was collected. After ultrafiltration, the buffer was replaced with PBS to obtain the endoglycosidase P1 modified with an azide group at the C-terminus. HRMS [M+H]+ The reference value is 31970.9 and the measured value is 31971.0.

[0077] Example 2: Preparation of protein C-terminal derivative P1

[0078]

[0079] The CPD-labeled mutant endoglycosidase Endo-F3 D165A-CPD, 3-azidopropylamine, 4-dimethylaminopyridine (DMAP) and phosphoinositide (InsP6) were dissolved in 50 mM sodium acetate solution (pH = 7.4) to make the concentrations of each component 100 μM, 5 mM, 5 mM, and 10 μM, respectively, and Ni-NTA agarose resin was added. The mixture was incubated at 4 ° C for 24 h, filtered, and the filtrate was collected. After ultrafiltration, the buffer was replaced with PBS to obtain the endoglycosidase P1 modified with an azide group at the C-terminus. HRMS [M+H] + The reference value is 31970.9 and the measured value is 31971.0.

[0080] Example 3: Preparation of protein C-terminal derivative P2

[0081]

[0082] The preparation method is the same as that of Example 1, except that 3-azidopropylamine is replaced with 1-amino-2-methyl-2-propanol, and the endoglycosidase P2 with an alcohol hydroxyl group modified at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 31959.9 and the measured value is 31960.0.

[0083] Example 4: Preparation of protein C-terminal derivative P3

[0084]

[0085] The preparation method is the same as that in Example 1, except that 3-azidopropylamine is replaced with propargylamine, and the endoglycosidase P3 modified with an alkynyl group at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 31925.8 and the measured value is 31926.0.

[0086] Example 5: Preparation of protein C-terminal derivative P4

[0087]

[0088] The preparation method is the same as that of Example 1, except that 3-azidopropylamine is replaced with glycine, and the endoglycosidase P4 modified with glycine at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 31945.8 and the measured value is 31946.0.

[0089] Example 6: Preparation of protein C-terminal derivative P5

[0090]

[0091] The preparation method is the same as that in Example 1, except that 3-azidopropylamine is replaced with glycine-glycine, and the endoglycosidase P5 modified with glycine-glycine at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 32002.9 and the measured value is 32003.0.

[0092] Example 7: Preparation of protein C-terminal derivative P6

[0093]

[0094] The preparation method is the same as that in Example 1, except that 3-azidopropylamine is replaced with 3-aminopropanesulfonic acid, and the endoglycosidase P6 with sulfonic acid modified at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 32009.9 and the measured value is 32010.0.

[0095] Example 8: Preparation of protein C-terminal derivative P7

[0096]

[0097] The preparation method is the same as that of Example 1, except that 3-azidopropylamine is replaced with 4-amino-1-butanol, and the endoglycosidase P7 with an alcohol hydroxyl group modified at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 31959.9 and the measured value is 31960.0.

[0098] Example 9: Preparation of protein C-terminal derivative P8

[0099]

[0100] The preparation method is the same as that in Example 1, except that 3-azidopropylamine is replaced with aminoethanethiol, and the endoglycosidase P8 with a thiol group modified at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 31947.9 and the measured value is 31948.0.

[0101] Example 10: Preparation of protein C-terminal derivative P9

[0102]

[0103] The preparation method is the same as that in Example 1, except that 3-azidopropylamine is replaced with ethylenediamine, and the endoglycosidase P9 modified with an amino group at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 31930.9 and the measured value is 31931.0.

[0104] Example 11: Preparation of protein C-terminal derivative P10

[0105]

[0106] The preparation method is the same as that of Example 1, except that 3-azidopropylamine is replaced with aminobiotin, and the endoglycosidase P10 modified with biotin at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 32157.1 and the measured value is 32157.0.

[0107] Example 12: Preparation of protein C-terminal derivative P11

[0108]

[0109] The preparation method is the same as that of Example 1, except that 3-azidopropylamine is replaced with propynyl-triethylene glycol-amino, and the endoglycosidase P11 with triethylene glycol-amino modified at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 32058.0, and the measured value is 32058.0.

[0110] Example 13: Preparation of protein C-terminal derivative P12

[0111]

[0112] The preparation method is the same as that in Example 1, except that 3-azidopropylamine is replaced with glycine-glycine-glycine, and the endoglycosidase P12 with glycine-glycine-glycine modified at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 32059.9 and the measured value is 32060.0.

[0113] Example 14: Preparation of protein C-terminal derivative P13

[0114]

[0115] The preparation method is the same as that in Example 1, except that 3-azidopropylamine is replaced with amino-diethylene glycol-azide, and the endoglycosidase P13 modified with diethylene glycol-azide at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 32045.0, and the measured value is 32045.0.

[0116] Example 15: Preparation of protein C-terminal derivative P14

[0117]

[0118] The preparation method is the same as that in Example 1, except that 3-azidopropylamine is replaced with hydrazine hydrate, and the endoglycosidase P14 modified with hydrazine at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 31902.8 and the measured value is 31903.0.

[0119] Example 16: Preparation of protein C-terminal derivative P15

[0120]

[0121] The preparation method is the same as that in Example 1, except that the CPD-labeled endoglycosidase Endo-F3 D165A-CPD is replaced with the CPD-labeled Her2 Nanobody-CPD (SEQ ID No.: 23), and the Her2 Nanobody P15 modified with an azide group at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 13352.0 and the measured value is 13351.0.

[0122] Example 17: Preparation of protein C-terminal derivative P16

[0123]

[0124] The preparation method is the same as that of Example 14, except that the CPD-labeled endoglycosidase Endo-F3 D165A-CPD is replaced with the CPD-labeled Her2 Nanobody-CPD, and the Her2 Nanobody P16 modified with hydrazine at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 13283.9 and the measured value is 13284.0.

[0125] Example 18: Preparation of protein C-terminal derivative P17

[0126]

[0127] The preparation method is the same as that of Example 1, except that the CPD-labeled endoglycosidase Endo-F3 D165A-CPD is replaced with the CPD-labeled green fluorescent protein GFP-CPD (SEQ ID No.: 24), and the green fluorescent protein P17 modified with an azide group at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 26173.1 and the measured value is 26173.0.

[0128] Example 19: Preparation of protein C-terminal derivative P18

[0129]

[0130] The preparation method is the same as that in Example 1, except that the CPD-labeled endoglycosidase Endo-F3 D165A-CPD is replaced with the CPD-labeled endoglycosidase Endo-A-CPD (SEQ ID No.: 25), and the endoglycosidase P18 modified with an azide group at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 70177.2 and the measured value is 70106.0.

[0131] Example 20: Preparation of protein C-terminal derivative P19

[0132]

[0133] The preparation method is the same as that of Example 1, except that the CPD-labeled endoglycosidase Endo-F3 D165A-CPD is replaced with the CPD-labeled glutathione S-transferase GST-CPD (SEQ ID No.: 26), and the glutathione S-transferase P19 modified with an azide group at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 25979.0 and the measured value is 25978.0.

[0134] Example 21: Preparation of protein C-terminal derivative P20

[0135]

[0136] The preparation method is the same as that in Example 1, except that the CPD-labeled endoglycosidase Endo-F3 D165A-CPD is replaced with the CPD-labeled endoglycosidase Endo-S2-CPD (SEQ ID No.: 27), and the endoglycosidase P20 modified with an azide group at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 86204.3 and the measured value is 86207.0.

[0137] Example 22: Preparation of protein C-terminal derivative P21

[0138]

[0139] The CPD-labeled mutant endoglycosidase Endo-F3 D165A-CPD, DBCO-NH2, 4-dimethylaminopyridine and phosphoinositide (InsP6) were dissolved in 50mM sodium acetate solution (pH=7.4) to make the concentrations of each item 100μM, 10mM, 5mM, and 20μM, respectively. After incubation at 4℃ for 5h, Ni-NTA agarose resin was added and incubated at 4℃ for 2h. The His-tagged CPD was bound, filtered, and the filtrate was collected. After ultrafiltration, the buffer was replaced with PBS to obtain the C-terminally modified DBCO endoglycosidase P21. HRMS[M+H] + The reference value is 32273.9 and the measured value is 32274.0.

[0140] Example 23: Preparation of protein C-terminal derivative P22

[0141]

[0142] The preparation method is the same as that of Example 1, except that 3-azidopropylamine is replaced with cystamine, and the endoglycosidase P22 modified with cystamine at the C-terminus is obtained after purification. HRMS [M+H] + The reference value is 32022.0, and the measured value is 32022.0.

[0143] Example 24: Preparation of protein C-terminal derivative P23

[0144]

[0145] The Her2 nanoantibody P15 with azide group modified at the C-terminus and DBCO-FITC were dissolved in 50mM PB buffer (pH=7.4) to a final concentration of 100μM and 500μM, respectively, and reacted at 37℃ for 2h to obtain the Her2 nanoantibody P23 with fluorescent group FITC modified at the C-terminus. HRMS[M+H] + The reference value is 14017.7 and the measured value is 14017.0.

[0146] Example 25: Preparation of protein C-terminal derivative P24

[0147]

[0148]

[0149] The Her2 nanoantibody P15 with azide group modified at the C-terminus was linked to the drug linker BCN-Lys (PEG 24)-VC-PAB-MMAE was dissolved in 50 mM PB buffer (pH = 7.4) to a final concentration of 100 μM and 500 μM, respectively, and reacted at 37°C overnight to obtain Her2 nanoantibody drug conjugate P24 with a drug linker coupled to the C-terminus. HRMS [M+H] + The reference value is 15923.1 and the measured value is 15923.0.

[0150] Example 26: Preparation of protein C-terminal derivative P25

[0151]

[0152] The Her2 nanoantibody P16 with hydrazide modified at the C-terminus was dissolved with sodium nitrite in 50mM glycine buffer (pH=3.0) to a final concentration of 50μM and 300μM, respectively. After reacting on ice for 30 minutes, 4-mercaptophenylacetic acid MPAA was added to a final concentration of 20mM. 500mM sodium hydroxide was used to adjust the pH to 6-6.5. The reaction was continued on ice for 15 minutes. Cysteine ​​was added to a final concentration of 50mM. 500mM sodium hydroxide was used to adjust the pH to 7-7.5. The reaction was carried out at room temperature for 1h to obtain the nanoantibody P25 with cysteine ​​coupled to the C-terminus. HRMS [M+H] + The reference value is 13373.0, and the measured value is 13373.0.

[0153] Example 27: Preparation of protein C-terminal derivative P26

[0154]

[0155] The Her2 nanoantibody P16 with hydrazide modified at the C-terminus was dissolved with sodium nitrite in 50mM glycine buffer (pH=3.0) to a final concentration of 50μM and 300μM, respectively. After reacting on ice for 30 minutes, 4-mercaptophenylacetic acid MPAA was added to a final concentration of 20mM. 500mM sodium hydroxide was used to adjust the pH to 6-6.5. The reaction was continued on ice for 15 minutes. The peptide H-CDYKDDDDK-OH was added to a final concentration of 50mM. 500mM sodium hydroxide was used to adjust the pH to 7-7.5. The reaction was carried out at room temperature for 5h to obtain the nanoantibody P26 with a Flag tag coupled to the C-terminus. HRMS [M+H] + The reference value is 14534.2 and the measured value is 14534.0.

[0156] Example 28: Preparation of protein C-terminal derivative P27

[0157]

[0158] The Her2 nanoantibody P16 with hydrazide modified at the C-terminus and N-acetylglucosamine GlcNAc were dissolved in 100mM sodium acetate buffer (pH=4.0) to a final concentration of 50μM and 1M, respectively, and reacted at 37°C overnight to obtain the nanoantibody P27 with N-acetylglucosamine coupled to the C-terminus. HRMS [M+H] + The reference value is 13487.1 and the measured value is 13487.0.

[0159] Example 29: Preparation of protein C-terminal derivative P28

[0160]

[0161] The preparation method is the same as that of Example 26, except that the Her2 nanoantibody P16 with hydrazide modified at the C-terminus is replaced with endoglycosidase P14 with hydrazide modified at the C-terminus, thereby obtaining endoglycosidase P28 with cysteine ​​modified at the C-terminus. HRMS [M+H] + The reference value is 31904.0, and the measured value is 31904.0.

[0162] Example 30: Preparation of protein C-terminal derivative P29

[0163]

[0164] The preparation method is the same as that of Example 27, except that the Her2 nanoantibody P16 with hydrazide modified at the C-terminus is replaced with endoglycosidase P14 with hydrazide modified at the C-terminus, thereby obtaining endoglycosidase P29 with a Flag tag modified at the C-terminus. HRMS [M+H] + The reference value is 33150.0, and the measured value is 33150.0.

[0165] Example 31: Preparation of protein C-terminal derivative P30

[0166]

[0167] The preparation method is the same as that of Example 28, except that the Her2 nanoantibody P16 with hydrazide modified at the C-terminus is replaced with endoglycosidase P14 with hydrazide modified at the C-terminus, thereby obtaining endoglycosidase P30 with N-acetylglucosamine modified at the C-terminus. HRMS [M+H] + The reference value is 32081.0, and the measured value is 32081.0.

[0168] Example 32: Preparation of protein C-terminal derivative P31

[0169]

[0170] The glycosidase P1 with modified azide group at the C-terminus and the drug linker DBCO-SMCC-DM1 were dissolved in 50mM PB buffer (pH=7.4) to a final concentration of 100μM and 500μM, respectively, and reacted at 37℃ for 2h to obtain the glycosidase P31 with drug linker coupled to the C-terminus. HRMS[M+H] + The reference value is 33205.1 and the measured value is 33205.0.

[0171] Example 33: Cysteine ​​protease-mediated hydrolysis of protein C-terminal derivative P1

[0172]

[0173] The C-terminally modified azide group endoglycosidase P1, cysteine ​​protease domain CPD, and inositol hexaphosphate InsP6 were dissolved in 1×PBS buffer (pH=7.4) to a final concentration of 40μM, 80μM, and 80μM, respectively, and reacted at room temperature for 12h to obtain the C-terminally unmodified endoglycosidase. HRMS [M+H] + The reference value is 31888.8 and the measured value is 31888.0.

[0174] Example 34: Cysteine ​​protease-mediated exchange of protein C-terminal functional groups (conversion of hydrazine to azide group)

[0175]

[0176] The Her2 nanoantibody P16 modified with hydrazide at the C-terminus was dissolved in sodium acetate buffer (pH=7.4) with 3-azidopropylamine, DMAP, cysteine ​​protease domain CPD, and inositol hexaphosphate InsP6 to a final concentration of 20μM, 5mM, 5mM, 20μM, and 10μM, respectively, and reacted at 4°C for 5h to obtain Her2 nanoantibody P15 modified with azide at the C-terminus. HRMS [M+H] + The reference value is 13352.0 and the measured value is 13351.0.

[0177] Example 35: Cysteine ​​protease-mediated exchange of protein C-terminal functional groups (conversion of azide groups to alkyne groups)

[0178]

[0179] The Her2 nanoantibody P15 modified with an azide group at the C-terminus was dissolved in sodium acetate buffer (pH = 7.4) with propargylamine, DMAP, cysteine ​​protease domain CPD, and inositol hexaphosphate InsP6 to a final concentration of 20 μM, 5 mM, 5 mM, 20 μM, and 10 μM, respectively, and reacted at 4°C for 10 h to obtain the Her2 nanoantibody P32 modified with an alkynyl group at the C-terminus. HRMS [M+H]+ The reference value is 13308.0, and the measured value is 13308.0.

[0180] Example 36: Preparation of dual antibody P32

[0181]

[0182] The Her2 nanoantibody P15 modified with an azide group at the C-terminus and the trastuzumab modified with DBCO at K251 (antibody modification products previously reported by the inventor of the present application, Angew. Chem. Int. Ed., 2022 Sep 5; 61(36): e202204132. doi: 10.1002 / anie.202204132.) were dissolved in PB buffer solution to a final concentration of 133 μM and 5 mg / mL, respectively. The reaction was carried out at 37° C. for 8 h to obtain the dual-epitope dual-antibody P32. After SDS-PAGE monitoring, the significant band appearing near 70 kDa was the heavy chain of the antibody coupled to the Her2 nanoantibody P15, indicating the synthesis of the target protein.

[0183] The amino acid and nucleotide sequences of the fusion proteins mentioned above are listed here, where the terminal HHHHHH is a purification tag, and may also be other purification tags known in the art:

[0184] Cysteine ​​protease domain CPD amino acid sequence (SEQ ID No.: 21)

[0185] ILHNQNVNSWGPITVTPTTDGGETRFDGQIIVQMENDPVVAKAAANLAGKHAESSVVVQLDSDGNYRVVYGDPSKLDGKLRWQLVGHGRDHSETNNTRLSGYSADE LAVKLAKFQQSFNQAENINNKPDHISIVGCSLVSDDKQKGFGHQFINAMDANGLRVDVSVRSSELAVDEAGRKHTKDANGDWVQKAENNKVSLSWDAQGLEHHHHHH

[0186] Endo-F3(D165A)-CPD amino acid sequence (SEQ ID No.: 22)

[0187] MATALAGSNGVCIAYYITDGRNPTFKLKDIPDKVDMVILFGLKYWSLQDTTKLPGGTGMMGSFKSYKDLDTQIRSLQSRGIKVLQNIDDDVSWQSSKPGGFASAAAYGDAIKSIVIDKWKLDGISLAIEHSGAKPNPIPTFPGYAATGYNGWYSGSMAATPAFLNVISELTKYFGTTAPNNKQLQIASGIDVYAWNKIMENFRNNFNYIQLQSYGANVSRTQLMMNYATGTNKIPASKMVFGAYAEGGTNQANDVEVAKWTPTQGAKGGMMIYTYNSNVSYANAVRDAVKNVDALADGKILHNQNVNSWGPITVTPTTDGGETRFDGQIIVQMENDPVVAKAAANLAGKHAESSVVVQLDSDGNYRVVYGDPSKLDGKLRWQLVGHGRDHSETNNTRLSGYSADELAVKLAKFQQSFNQAENINNKPDHISIVGCSLVSDDKQKGFGHQFINAMDANGLRVDVSVRSSELAVDEAGRKHTKDANGDWVQKAENNKVSLSWDAQGLEHHHHHH

[0188] CPD-labeled Her2 nanobody Her2 Nb-CPD amino acid sequence (SEQ ID No.: 23)

[0189] MQVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVSSGGVDALADGKILHNQNVNSWGPITVTPTTDGGETRFDGQIIVQMENDPVVAKAAANLAGKHAESSVVVQLDSDGNYRVVYGDPSKLDGKLRWQLVGHGRDHSETNNTRLSGYSADELAVKLAKFQQSFNQAENINNKPDHISIVGCSLVSDDKQKGFGHQFINAMDANGLRVDVSVRSSELAVDEAGRKHTKDANGDWVQKAENNKVSLSWDAQGLEHHHHHH

[0190] Amino acid sequence of GFP-CPD labeled with CPD (SEQ ID No.: 24)

[0191] MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRYEAGRVIGDFKVMGTGFPEDSVIFTDKIIRSNATVEHLHP

[0192] MGDNDLDGSFTRTFSLRDGGYYSSVVDSHMHFKSAIHPSILQNGGPMFAF

[0193] RRVEEDHSNTELGIVEYQHAFKTPDADAGEEVDALADGKILHNQNVNSW

[0194] GPITVTPTTDGGETRFDGQIIVQMENDPVVAKAAANLAGKHAESSVVVQL

[0195] DSDGNYRVVYGDPSKLDGKLRWQLVGHGRDHSETNNTRLSGYSADELA

[0196] VKLAKFQQSFNQAENINNKPDHISIVGCSLVSDDKQKGFGHQFINAMDAN

[0197] GLRVDVSVRSSELAVDEAGRKHTKDANGDWVQKAENNKVSLSWDAQG

[0198] LEHHHHHH

[0199] Amino acid sequence of Endo-A-CPD labeled with CPD (SEQ ID No.: 25)

[0200] MSTYNGPLSSHWFPEELAQWEPDSDPDAPFNRSHVPLEPGRVANRVNAN

[0201] ADKDAHLVSLSALNRHTSGVPSQGAPVFYENTFSYWHYTDLMVYWAGS

[0202] AGEGIIVPPSADVIDASHRNGVPILGNVFFPPTVYGGQLEWLEQMLEQEED

[0203] GSFPLADKLLEVADYYGFDGWFINQETEGADEGTAEAMQAFLVYLQEQK

[0204] PEGMHIMWYDSMIDTGAIAWQNHLTDRNKMYLQNGSTRVADSMFLNFW

[0205] WRDQRQSNELAQALGRSPYDLYAGVDVEARGTSTPVQWEGLFPEGEKA

[0206] HTSLGLYRPDWAFQSSETMEAFYEKELQFWVGSTGNPAETDGQSNWPGM

[0207] AHWFPAKSTATSVPFVTHFNTGSGAQFSAEGKTVSEQEWNNRSLQDVLPT

[0208] WRWIQHGGDLEATFSWEEAFEGGSSLQWHGSLAEGEHAQIELYQTELPIS

[0209] EGTSLTWTFKSEHGNDLNVGFRLDGEEDFRYVEGEQRESINGWTQWTLP

[0210] LDAFAGQTITGLAFAAEGNETGLAEFYIGQLAVGADSEKPAAPNVNVRQY

[0211] DPDPSGIQLVWEKQSNVHHYRVYKETKHGKELIGTSAGDRIYLEGLVEES

[0212] KQNDVRLHIEALSETFVPSDARMIDIKSGSFVDALADGKILHNQNVNSWG

[0213] PITVTPTTDGGETRFDGQIIVQMENDPVVAKAAANLAGKHAESSVVVQLD

[0214] SDGNYRVVYGDPSKLDGKLRWQLVGHGRDHSETNNTRLSGYSADELAV

[0215] KLAKFQQSFNQAENINNKPDHISIVGCSLVSDDKQKGFGHQFINAMDANG

[0216] LRVDVSVRSSELAVDEAGRKHTKDANGDWVQKAENNKVSLSWDAQGLE

[0217] HHHHHH

[0218] CPD-labeled glutathione S-transferase GST-CPD amino acid sequence (SEQ ID No.: 26)

[0219] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGL

[0220] EFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLD

[0221] IRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHP

[0222] DFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAW

[0223] PLQGWQATFGGGDHPPKVDALADGKILHNQNVNSWGPITVTPTTDGGET

[0224] RFDGQIIVQMENDPVVAKAAANLAGKHAESSVVVQLDSDGNYRVVYGD

[0225] PSKLDGKLRWQLVGHGRDHSETNNTRLSGYSADELAVKLAKFQQSFNQA

[0226] ENINNKPDHISIVGCSLVSDDKQKGFGHQFINAMDANGLRVDVSVRSSEL

[0227] AVDEAGRKHTKDANGDWVQKAENNKVSLSWDAQGLEHHHHHH

[0228] CPD-labeled endoglycosidase Endo-S2-CPD amino acid sequence (SEQ ID No.: 27)

[0229] MEKTVQTGKTDQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGK

[0230] QQHPENTMAEVPKEVDILFVFHDHTASDSPFWSELKDSYVHKLHQQGTA

[0231] LVQTIGVNELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDID

[0232] IEHEFTNKRTPEEDARALNVFKEIAQLIGKNGSDKSKLLIMDTTLSVENNPI

[0233] FKGIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQFMIGFSF

[0234] FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKAG

[0235] IFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKTLMTE

[0236] DKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGDKIQNLKG

[0237] LEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGMTGLEKLNLS

[0238] GLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKLLMTLMEQVSNHQ

[0239] KITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEHDILTDFVFGTVTK

[0240] RNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYAEAFRKDYKGYKVHLTASNL

[0241] GETVTSKVTATTDETYLVDVSDGEKVVHHMKLNIGSGAIMMENLAKGAK

[0242] IMPORTANT:

[0243] ETNTEIKTDSSLNVAKGRLQILKDTTIDLEKMDIKNRKEYLSNDENWTDV

[0244] AQMDDAKAVDALADGKILHNQNVNSWGPITVTPTTDGGETRFDGQIIVQ

[0245] MENDPVVAKAAANLAGKHAESSVVVQLDSDGNYRVVYGDPSKLDGKLR

[0246] WQLVGHGRDHSETNNTRLSGYSADELAVKLAKFQQSFNQAENINNKPDH

[0247] ISIVGCSLVSDDKQKGFGHQFINAMDANGLRVDVSVRSELAVDEAGRKH

[0248] TKDANGDWVQKAENNKVSLSWDAQGLEHHHHHH

Claims

1. A method for selectively modifying the C-terminus of a protein, the method comprising the following steps 1): A nucleophile, a catalyst and a cysteine ​​protease allosteric activator are added to the target protein of the cysteine ​​protease domain (CPD) fused with the following formula (I), and the C-terminally modified target protein is released while inducing the cleavage of the CPD: in, In formula (I), the cysteine ​​protease recognition sequence is an amino acid motif that can be recognized by the cysteine ​​protease domain, and its sequence is XXLXXXX, X is any amino acid, and L is leucine (Leu); preferably, its sequence is VDALADGK (SEQ ID No.: 1), KEALADGK (SEQ ID No.: 2), LHALGETS (SEQ ID No.: 3), LDALSGNE (SEQ ID No.: 4), KGSLSGAN (SEQ ID No.: 5), LEGLSGTN (SEQ ID No.: 6), LDWLSGKN (SEQ ID No.: 7), LQALSGKN (SEQ ID No.: 8), SGALMSQY (SEQ ID No.: 9), GAALADGK (SEQ ID No.: 10); PRT represents the target protein to be modified, which is an amino acid sequence comprising at least 3 amino acids in length; CPD is a cysteine ​​protease domain. Optionally, the method further comprises the step of separating and purifying the obtained C-terminally modified target protein.

2. The method according to claim 1, in, The PRT in formula (I) is derived from mutant endoglycosidase Endo-F3D165A, Her2 nanoantibody, green fluorescent protein GFP, endoglycosidase Endo-A, endoglycosidase Endo-S2 or glutathione S-transferase GST.

3. The method according to claim 1, in, The nucleophilic agent has a structure shown in the following formula (II): NH 2 -X-R (II) in, X is absent or is a linking group selected from an unsubstituted or substituted alkylene group, a polyethylene glycol group, wherein the carbon in the alkylene backbone may be optionally replaced by S, N heteroatoms; and preferably, the substituent is selected from (=O), C1-C6 alkyl; R is a reactive group that can be further reacted or modified, a biologically active group, or a detectable reactive group; Preferably, R is a bioorthogonal group; Preferably, R is selected from the following groups: an azide group, an amino group, an alkynyl group, an amino group, a hydroxyl group, a thiol group, a sulfonic acid group, and a trans-cyclooctene group; Preferably, R is a group derived from an amino acid, a peptide chain, a protein, a sugar, a fluorescent molecule, a metal ion, or a nucleotide; Preferably, R is a group derived from a drug or a drug-linker, for example, the drug is a tumor therapeutic drug; Preferably, R is a group derived from a fluorescent molecule.

4. The method according to claim 3, in, The C-terminally modified target protein obtained by the method has a structure shown in the following formula (III): wherein PRT, X, and R are defined as in claim 1 or 3, respectively; The cysteine ​​protease recognition sequence 1 is a truncated sequence of the cysteine ​​protease recognition sequence in formula (I), having the general formula XXXL, wherein X is any amino acid and L is leucine (Leu); preferably, the cysteine ​​protease recognition sequence 1 is VDAL (SEQ ID No.: 11), LDAL (SEQ ID No.: 12), LHAL (SEQ ID No.: 13), KGSL (SEQ ID No.: 14), LEGL (SEQ ID No.: 15), LDWL (SEQ ID No.: 16), LQAL (SEQ ID No.: 17), SGAL (SEQ ID No.: 18), GAAL (SEQ ID No.: 19) or KEAL (SEQ ID No.: 20).

5. The method according to claim 1, in, The nucleophile is selected from any one of the following: and / or The catalyst is selected from any one of the following: and / or The cysteine ​​protease allosteric activator is selected from inositol hexaphosphate (InsP 6 ) and its hydrolyzate, preferably, selected from any one of the following:

6. The method according to claim 4, in, The method further comprises a hydrolysis step 2) after step 1): The C-terminally modified target protein of formula (III) is reacted with cysteine ​​protease CPD and a cysteine ​​protease allosteric activator to obtain a hydrolyzate of formula (III), which has a structure shown in the following formula (IV): PRT-XXXL-COOH(IV) Wherein, X is any amino acid, L is leucine, Preferably, the hydrolyzate of formula (III) has the following structure: PRT-VDAL-COOH; Preferably, in step 2), the final concentration of cysteine ​​protease in the reaction system is 2 μM-500 μM, and the final concentration of cysteine ​​protease allosteric activator is 2 μM-500 μM; Preferably, in step 2), the pH of the reaction system is 7.4-8.5, and the buffer is selected from phosphate buffer, sodium acetate buffer, Tris-HCl buffer, PIPES buffer, MES buffer, HEPES buffer, and BES buffer; Preferably, in step 2), the reaction temperature is 4°C-37°C, and the reaction time is 10 min-10 h.

7. The method according to claim 4, in, The method further comprises a step 3) of converting the protein C-terminal modification after step 1): In the presence or absence of a catalyst, the C-terminally modified target protein of formula (III) is reacted with other nucleophiles, cysteine ​​protease CPD and cysteine ​​protease allosteric activator to obtain a target protein having a C-terminally modified structure different from the modified structure of step 1), wherein the other nucleophiles refer to nucleophiles different from the nucleophiles used in step 1), Preferably, in step 3), there is no need to perform side chain protection on the protein to be modified; Preferably, in the step 3), in the reaction system, the final concentration of other nucleophiles is 2mM-100mM, the final concentration of the catalyst is 0-10mM, the final concentration of cysteine ​​protease is 2μM-500μM, and the final concentration of the cysteine ​​protease allosteric activator is 2μM-500μM; Preferably, in the step 3), the pH of the reaction system is 7.4-8.5, and the buffer is selected from phosphate buffer, sodium acetate buffer, Tris-HCl buffer, PIPES buffer, MES buffer, HEPES buffer, BES buffer, preferably, the buffer is PBS phosphate buffer, sodium acetate buffer or MES buffer; Preferably, in step 3), the reaction temperature is 4°C-37°C, and the reaction time is 10 min-10 h.

8. The method according to claim 6 or 7, in, The cysteine ​​protease allosteric activator used in steps 2) and 3), the other nucleophilic reagent and the catalyst used in step 3) are as defined in claim 5, respectively.

9. The method according to claim 4, in, When R in the C-terminally modified target protein of formula (III) is a bioorthogonal group, the method further includes a secondary modification step: further reacting the C-terminally modified target protein of formula (III) with a functional molecule or a pharmaceutically active molecule to obtain a product in which the C-terminus of the target protein is secondary modified.

10. A C-terminally modified target protein obtained by the method according to any one of claims 1 to 10.

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