Composition comprising copper-transport protein-fused tyrosinase

Abstract

To provide a technology that enables efficient production of an organic compound-modified polypeptide by means of copper-transport protein-fused tyrosinase.SOLUTION: The copper-transport protein-fused tyrosinase is brought into contact with a target polypeptide while the tyrosinase active site remains covered by the copper-transport protein without being cleaved by a protease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a composition comprising a copper transport protein fusion tyrosinase, and a method for producing a modified polypeptide using a copper transport protein fusion tyrosinase. [Background technology]

[0002] DOPA (L-3,4-dihydroxyphenylalanine) is found in large amounts in the byssus proteins of shellfish (barnacles, mussels, etc.) and is known to exert excellent adhesive properties in water by forming chelates with metals, coupling with other DOPA molecules, and forming covalent bonds with nucleophiles on the surface of organic tissues. DOPA residues in proteins are formed by the oxidation of tyrosine residues in byssus proteins by tyrosinase. Furthermore, quinone crosslinking by DOPAquinone, formed by further oxidation of DOPA by tyrosinase, has also been reported to be involved in adhesion. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Chen Y, Loredo A, Chung A, Zhang M, Liu R, Xiao H. Biosynthesis and Genetic Incorporation of 3,4-Dihydroxy-L-Phenylalanine into Proteins in Escherichia coli. J Mol Biol. 2022 Apr 30;434(8):167412. doi: 10.1016 / j.jmb.2021.167412. Summary of the Invention [Problem to be solved by the invention]

[0004] The inventors previously developed a technology that uses a fusion tyrosinase in which tyrosinase and a copper transport protein are linked via a linker sequence and a protease recognition sequence to turn on tyrosinase activity at any time, converting tyrosine residues in a protein to DOPA residues or DOPAquinone residues and imparting adhesive properties to the protein. More specifically, the technology is as follows: When tyrosinase is linked to a copper transport protein, the tyrosinase activity is inhibited because the tyrosinase active site is shielded by the copper transport protein. On the other hand, if the protease recognition sequence in the fusion tyrosinase is cleaved by a protease, the copper transport protein is separated from the tyrosinase, exposing the tyrosinase active site. Based on this mechanism of action, the inventors previously developed a technology that can impart adhesive properties to the protein by adding a protease to release the inhibition of tyrosinase activity at any time.

[0005] Proteins containing DOPA residues or DOPAquinone residues are known to have various functions other than adhesive properties. For example, Non-Patent Document 1 (2013) studies a technique for using a protein containing DOPA residues to induce a strain-promoted oxidation-controlled cyclooctyne-1,2-quinone (SPOCQ) cycloaddition reaction at the DOPA residue. In the technique described in Non-Patent Document 1, proteins containing DOPA residues are prepared using Escherichia coli modified to translate proteins containing DOPA residues at any position using an amber codon. More specifically, proteins containing DOPA residues at any position are prepared using Escherichia coli genetically modified to recognize the amber codon (UAG), which is normally recognized as a stop codon, as the codon corresponding to DOPA. That is, in Non-Patent Document 1, proteins containing DOPA residues are prepared by incorporating DOPA into a protein during synthesis at the translation stage.

[0006] Although the technology in Non-Patent Document 1 has the advantage of being able to place DOPA residues at any position in the protein structure, there were concerns that the yield of the target protein would be lower than that of a conventional protein synthesis system using E. coli because an amber codon, which is normally a stop codon, is used for DOPA incorporation.

[0007] In light of the above, the present inventors attempted to convert tyrosine residues in translated polypeptides to DOPA residues or DOPAquinone residues using copper transport protein-fused tyrosinase and then attach other organic substances via the DOPA or DOPAquinone residues. However, when protease was added to a reaction system containing copper transport protein-fused tyrosinase, a target polypeptide (a polypeptide to which another organic substance is to be attached), and the target organic substance to derepress tyrosinase activity, the adhesive properties of the resulting DOPA or DOPAquinone residues resulted in the formation of complexes between the target polypeptide and protease, as well as complexes between the target polypeptides themselves, and the production of the intended organic substance-modified polypeptide was not confirmed (Figure 7). Therefore, the present inventors conducted extensive research, primarily with the goal of providing a technology for efficiently producing organic substance-modified polypeptides using copper transport protein-fused tyrosinase. [Means for solving the problem]

[0008] Unexpectedly, when no protease was added to a reaction system containing copper transport protein-fused tyrosinase, a target polypeptide, and the organic substance to be added, the production of the target organic substance-modified polypeptide was confirmed (Figure 7). That is, the present inventors discovered that by contacting copper transport protein-fused tyrosinase with the target polypeptide while the tyrosinase active site remains shielded by the copper transport protein, without cleaving it with a protease, it is possible to obtain an organic substance-modified polypeptide while suppressing the formation of a complex between the target polypeptides. The present inventors made further improvements and have now completed the present disclosure.

[0009] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. A composition comprising a copper transport protein fusion tyrosinase, The copper transport protein fusion tyrosinase is Sequence (A): an amino acid sequence having copper transport protein function; Sequence (B): a linker sequence, and Sequence (C): Amino acid sequence with tyrosinase activity and the sequences (A) to (C) are linked in the order of (A)-(B)-(C), A composition for use in contacting the copper transport protein fusion tyrosinase with a polypeptide of interest that contains a tyrosine residue. Section 2. A composition for producing a modified polypeptide via said contacting, comprising: the modified polypeptide comprises a structure in which a DOPA residue and / or a DOPA quinone residue obtained by converting the tyrosine residue is bound to an organic substance having a reactive functional group, Item 2. The composition according to Item 1, wherein the reactive functional group is a functional group reactive with a DOPA residue and / or a DOPA quinone residue. Section 3. Item 3. The composition according to Item 1 or 2, wherein the sequence (A) and / or the sequence (C) is an amino acid sequence derived from a bacterium of the genus Streptomyces. Section 4. The sequence (A) is Sequence (a1): the amino acid sequence represented by SEQ ID NO: 1, or Sequence (a2): The composition according to any one of Items 1 to 3, wherein the sequence identity to the amino acid sequence represented by SEQ ID NO:1 is 70% or more. Section 5. The sequence (C) is Sequence (c1): the amino acid sequence represented by SEQ ID NO: 2, or Sequence (c2): The composition according to any one of Items 1 to 4, which is an amino acid sequence having a sequence identity of 70% or more to the amino acid sequence represented by SEQ ID NO:2. Section 6. The sequence (A) is Sequence (a1): the amino acid sequence represented by SEQ ID NO: 1, or Sequence (a2): an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence represented by SEQ ID NO: 1, and The sequence (C) is Sequence (c1): the amino acid sequence represented by SEQ ID NO: 2, or Sequence (c2): The composition according to any one of Items 1 to 5, which is an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence represented by SEQ ID NO:2. Section 7. Item 7. The composition according to any one of Items 1 to 6, wherein the sequence (B) comprises at least one sequence selected from the group consisting of a GS linker sequence and a protease recognition sequence. Section 8. The copper transport protein fusion tyrosinase is Sequence (d1): the amino acid sequence represented by SEQ ID NO: 5, or Sequence (d2): an amino acid sequence having a sequence identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 5 Item 8. The composition according to any one of Items 1 to 7, comprising: Section 9. Item 9. The composition according to any one of Items 1 to 8, wherein the target polypeptide contains a tyrosine residue within 20 amino acid residues from the N-terminus and / or within 20 amino acid residues from the C-terminus. Section 10. The polypeptide of interest has at its N-terminus and / or C-terminus (GSHY) n Item 10. The composition according to any one of Items 1 to 9, comprising an amino acid sequence represented by the formula (n is a natural number of 1 to 10). Section 11. The reactive functional group is an amino group, a thiol group, an imidazole group, an alkynyl group, or a group represented by the following general formula (I): [ka] 11. The composition according to any one of items 2 to 10, wherein the group is at least one selected from the group consisting of groups represented by the following formula: Section 12. Item 12. The composition according to any one of Items 1 to 11, which is used for producing a bioconjugate. Section 13. Item 13. The composition according to any one of Items 1 to 12, wherein the target polypeptide is an antibody or an antibody fragment. Section 14. A method for producing a modified polypeptide, comprising: contacting a copper transport protein fusion tyrosinase with a polypeptide of interest to convert at least one tyrosine residue contained in the polypeptide of interest into a DOPA residue and / or a DOPA quinone residue; The copper transport protein fused tyrosinase Sequence (A): an amino acid sequence having copper transport protein function; Sequence (B): a linker sequence, and Sequence (C): Amino acid sequence with tyrosinase activity and the sequences (A) to (C) are linked in the order of (A)-(B)-(C). Section 15. the modified polypeptide comprises a structure in which a DOPA residue and / or a DOPA quinone residue obtained by converting the tyrosine residue is bound to an organic substance having a reactive functional group, Item 15. The composition according to Item 14, wherein the reactive functional group is a functional group reactive with a DOPA residue and / or a DOPA quinone residue. Section 16. Item 16. The production method according to Item 14 or 15, wherein the sequence (A) and / or the sequence (C) is an amino acid sequence derived from a bacterium of the genus Streptomyces. Section 17. The sequence (A) is Sequence (a1): the amino acid sequence represented by SEQ ID NO: 1, or 17. The method according to any one of Items 14 to 16, wherein sequence (a2) is an amino acid sequence having a sequence identity of 70% or more to the amino acid sequence represented by SEQ ID NO:1. Section 18. The sequence (C) is Sequence (c1): the amino acid sequence represented by SEQ ID NO: 2, or 18. The method according to any one of Items 14 to 17, wherein sequence (c2) is an amino acid sequence having a sequence identity of 70% or more to the amino acid sequence represented by SEQ ID NO:2. Section 19. The sequence (A) is Sequence (a1): the amino acid sequence represented by SEQ ID NO: 1, or Sequence (a2): an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence represented by SEQ ID NO: 1, and The sequence (C) is Sequence (c1): the amino acid sequence represented by SEQ ID NO: 2, or 19. The method according to any one of Items 14 to 18, wherein sequence (c2) is an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence represented by SEQ ID NO:2. Section 20. 20. The production method according to any one of Items 14 to 19, wherein the sequence (B) comprises at least one sequence selected from the group consisting of a GS linker sequence and a protease recognition sequence. Section 21. The copper transport protein fusion tyrosinase is Sequence (d1): the amino acid sequence represented by SEQ ID NO: 5, or Sequence (d2): an amino acid sequence having a sequence identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 5 21. The method according to any one of items 14 to 20, comprising: Section 22. 22. The method according to any one of Items 14 to 21, wherein the target polypeptide contains a tyrosine residue within 20 amino acid residues from the N-terminus and / or within 20 amino acid residues from the C-terminus. Section 23. The polypeptide of interest has at its N-terminus and / or C-terminus (GSHY) n 23. The method according to any one of items 14 to 22, wherein the amino acid sequence is represented by the formula (n is a natural number of 1 to 10). Section 24. The reactive functional group is an amino group, a thiol group, an imidazole group, an alkynyl group, or a group represented by the following general formula (I): [ka] 24. The method according to any one of items 15 to 23, wherein the group is at least one selected from the group consisting of groups represented by the following formula: Section 25. A method for producing a bioconjugate, comprising the steps of any one of items 14 to 24. Section 26. 26. The method according to any one of Items 14 to 25, wherein the target polypeptide is an antibody. [Effects of the Invention]

[0010] The present disclosure provides a new application of copper transport protein-fused tyrosinase. More specifically, the present disclosure provides a technology for efficiently producing organic substance-modified polypeptides while suppressing the formation of polypeptide complexes due to the adhesive properties of DOPA residues and DOPA quinone residues. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows a model diagram of the copper transport protein-fused tyrosinase expression plasmid prepared in Test Example 1-1. [Figure 2] The results of Test Example 1-4 are shown below. From the left, the lanes are molecular weight markers, the soluble fraction, and the insoluble fraction. [Figure 3] The results of Test Example 1-5 are shown below. From the left, the lanes are molecular weight marker, pre-column, flow-through fraction, wash fractions 1-3, and elution fractions 1-3. [Figure 4] The three-dimensional structure of LgBiT-Y predicted by the protein structure prediction program AlphaFold2 (DeepMind) is shown. Tyrosine residues are shown in green. [Figure 5] The results of Test Example 2-1-1 are shown. Multiple bands with a molecular weight of 50,000 or more that were observed only in the rightmost lane, which contains all of the fusion tyrosinase, 3C Protease, and substrate (LgBiT-Y), are boxed. [Figure 6]The results of Test Example 2-1-2 are shown. The region of molecular weight of 50,000 or more where multiple bands were observed in Test Example 2-1-1 but no bands were observed in this test is shown boxed. [Figure 7] The results of Test Example 2-2-1 are shown below. The left image shows the result of CBB staining of the electrophoresis gel, and the right image shows the result of photographing the electrophoresis gel with an imager equipped with a CCD camera. [Figure 8] The results of Test Example 2-2-2 are shown below. The left image shows the result of CBB staining of the electrophoresis gel, and the right image shows the result of photographing the electrophoresis gel with an imager equipped with a CCD camera. [Figure 9] The results of Test Example 2-2-3 are shown below. [Figure 10] The results of Test Example 2-3-2 are shown below. The left image shows the result of CBB staining of the electrophoresis gel, and the right image shows the result of photographing the electrophoresis gel with an imager equipped with a CCD camera. [Figure 11] The results of Test Example 2-4 are shown below. The left image shows the result of CBB staining of the electrophoresis gel, and the right image shows the result of photographing the electrophoresis gel with an imager equipped with a CCD camera. [Figure 12] Illustrative amino acid sequences encompassed by the present disclosure are shown. [Figure 13] Examples of base sequences encompassed by the present disclosure are shown below. DETAILED DESCRIPTION OF THE INVENTION

[0012] Each embodiment included in the present disclosure will be described in more detail below. The present disclosure preferably includes, but is not limited to, a composition containing copper transport protein fusion tyrosinase and a method for producing a modified polypeptide using copper transport protein fusion tyrosinase. The present disclosure includes all of the disclosures herein that would be recognized by a person skilled in the art.

[0013] A composition comprising a copper transport protein fusion tyrosinase encompassed by the present disclosure comprises a copper transport protein fusion tyrosinase comprising sequence (A): an amino acid sequence having copper transport protein function, sequence (B): a linker sequence, and sequence (C): an amino acid sequence having tyrosinase activity, wherein sequences (A) to (C) are linked in the order (A)-(B)-(C). Such a composition encompassed by the present disclosure may be referred to as the "composition of the present disclosure." Furthermore, such a copper transport protein fusion tyrosinase contained in a composition of the present disclosure may be referred to as the "fusion tyrosinase of the present disclosure."

[0014] 1. Fusion tyrosinases of the present disclosure 1-1. Sequence (A): Amino acid sequence with copper transport protein function In the present disclosure, the term "copper transport protein" refers to a protein capable of transporting copper to tyrosinase. In the present disclosure, the term "amino acid sequence having the function of a copper transport protein" refers to an amino acid sequence capable of exerting the function of the copper transport protein. Proteins capable of transporting copper to tyrosinase are known, and examples thereof include copper transport proteins derived from the genus Streptomyces, copper transport proteins derived from the genus Rhodococcus, and copper transport proteins derived from Actinomycetes other than these genera.

[0015] The amino acid sequence having the function of a copper transport protein may be the amino acid sequence of a known copper transport protein itself, or may be an amino acid sequence in which one or more amino acid residues have been substituted, deleted, or added from the amino acid sequence of a known copper transport protein, as long as it is capable of transporting copper and can shield at least a portion of the active site of tyrosinase when fused with tyrosinase. In the present disclosure, the amino acid sequence having the function of a copper transport protein is preferably the amino acid sequence of a copper transport protein derived from the genus Streptomyces.

[0016] In the present disclosure, the term "residue (amino acid residue)" refers to a structural unit of a polypeptide, a divalent group formed by removing one hydrogen atom from one amino group (-NH2) and one hydroxyl group (-OH) from one carboxy group (-COOH) contained in an amino acid. In addition, in the present disclosure, the term "polypeptide" refers to a molecule having a structure in which multiple amino acids are linked by peptide bonds, and molecules generally referred to as "oligopeptides" and "proteins" are also encompassed by the term "polypeptide" in the present disclosure.

[0017] The amino acid may be any of α-amino acids, β-amino acids, γ-amino acids, etc., but is preferably an α-amino acid. When the amino acid is an α-amino acid, the amino group and carboxy group are preferably directly bonded to the α-carbon, but may also be an amino group and / or a carboxy group contained in a side chain.

[0018] When the amino acid is an α-amino acid in which the amino group and the carboxy group are directly bonded to the α-carbon, the amino acid residue is represented by the following general formula (II): [ka] (wherein R is a so-called "side chain" and is a hydrogen atom or any substituent). As can be seen from the above formula, except when R is a hydrogen atom, the carbon atom to which R is bonded is an asymmetric carbon atom. In the present disclosure, the amino acid residues constituting the polypeptide may be either L- or D-isomers, or may be a mixture of L- and D-isomers. In the present disclosure, it is preferred that the amino acid residues constituting the polypeptide do not contain D-isomers.

[0019] More specific examples of copper transport proteins derived from the genus Streptomyces include those described in Uniprot (The Universal Protein Resource) entry numbers P55046, P55047, P55048, P17687, etc. Other examples of copper transport proteins derived from actinomycetes include those described in Uniprot entry number A0A0N1NTI5, etc. Uniprot is a publicly known database that provides protein amino acid sequences and information on their functions. Amino acid sequences having copper transport protein functions can be easily determined based on databases such as Uniprot.

[0020] An example of an amino acid sequence (SEQ ID NO: 1) having copper transport protein function is shown in Figure 12. The amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence corresponding to the copper transport protein derived from the genus Streptomyces, described in Uniprot entry number P17687 (also known as ORF438, derived from Streptomyces antibioticus). In the present disclosure, the amino acid sequence having copper transport protein function can be used alone or in combination of two or more.

[0021] In the present disclosure, amino acid sequences having copper transport protein function include the amino acid sequence represented by SEQ ID NO: 1 and amino acid sequences having 70% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 1. The sequence identity can be 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0022] In the present disclosure, "identity" of amino acid sequences refers to the degree of amino acid sequence match between two or more comparable amino acid sequences. Therefore, the greater the match between two amino acid sequences, the greater the identity or similarity between those sequences. The level of amino acid sequence identity can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul S F. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul S F. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX has been developed based on this BLAST algorithm. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0023] Examples of amino acid sequences that do not have 100% sequence identity to a certain amino acid sequence X include amino acid sequences in which one or more amino acids have been substituted, deleted, added, or inserted (preferably by substitution, more preferably by conservative substitution) relative to the amino acid sequence X. Here, "multiple" means, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 or 3. The upper or lower limit of the range may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0024] "Conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine constitute conservative substitutions. Other examples of conservative substitutions include substitutions between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0025] As described above, in the present disclosure, a copper transport protein refers to a protein that can transport copper and, when fused with tyrosinase, can shield at least a portion of the active site of tyrosinase. Here, whether a protein can transport copper and, when fused with tyrosinase, can shield at least a portion of the active site of tyrosinase is confirmed by separating a fusion tyrosinase into the protein portion and the tyrosinase portion by protease treatment or the like, and finding that the tyrosinase activity is higher after separation than before separation. Methods for evaluating tyrosinase activity are well known. Furthermore, whether a protein can transport copper to tyrosinase and whether a protein can shield the active site of tyrosinase when fused with tyrosinase can also be predicted by computer simulation based on the amino acid sequence of the protein. Specific examples of computer simulation include structural model construction using AlphaFold2 (DeepMind) and analysis using the integrated computational chemistry system MOE (Chemical Computing Group).

[0026] 1-2. Sequence (B): Linker sequence In the present disclosure, the specific amino acid sequence of sequence (B): linker sequence is not particularly limited as long as the effects of the present disclosure are achieved. To this extent, the linker sequence may be any linker sequence known in the art or a linker sequence obtained by arbitrarily modifying a known linker sequence.

[0027] Specific examples of linker sequences include, but are not limited to, GS linker sequences consisting of glycine (G) and serine (S), polyglycine linker sequences, and protease recognition sequences. Examples of GS linker sequences include, for example, (GS) m A linker sequence represented by (G4S) m and a linker sequence represented by GS (G4S) mExamples of preferred GS linker sequences include linker sequences represented by SG. m can be, for example, a natural number of 1 to 20, preferably 1 to 10, and more preferably 2 to 5. The upper or lower limit of m can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. An example of a preferred GS linker sequence is GS(G4S)3SG (SEQ ID NO: 3).

[0028] Examples of protease recognition sequences include LEVLFQGP (SEQ ID NO: 4), LVPRGS (SEQ ID NO: 6), DDDDK (SEQ ID NO: 7), and IEGR (SEQ ID NO: 8). An example of a preferred protease recognition sequence is LEVLFQGP (SEQ ID NO: 4).

[0029] In the present disclosure, the linker sequence preferably contains at least one sequence selected from the group consisting of a GS linker sequence and a protease recognition sequence. It is more preferable that the linker sequence contains a GS linker sequence, and even more preferable that the linker sequence contains a GS linker sequence but does not contain a protease recognition sequence. More specifically, the linker sequence preferably contains, for example, GS(G4S)3SG (SEQ ID NO: 3) and / or LEVLFQGP (SEQ ID NO: 4), and more preferably contains GS(G4S)3SG (SEQ ID NO: 3).

[0030] As described above, in the technology of the present disclosure, the fusion tyrosinase is used without being cleaved by a protease, and therefore, in the present disclosure, the linker sequence does not need to include a protease recognition sequence.

[0031] In the present disclosure, the length of the linker sequence is not particularly limited as long as the effects of the present disclosure are achieved, and may be, for example, 1 to 200 amino acid residues. The upper or lower limit of the range is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, It may also be 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues. In the present disclosure, the length of the linker sequence is preferably 2 to 150 amino acid residues, more preferably 5 to 100 amino acid residues, and even more preferably 10 to 50 amino acid residues.

[0032] 1-3. Sequence (C): Amino acid sequence with tyrosinase activity In the present disclosure, the term "tyrosinase activity" refers to the activity of tyrosinase. Tyrosinase is a type III copper protein found in a variety of bacteria, fungi, plants, insects, crustaceans, mammals, etc., and its EC number is 1.14.18.1. It is known to be involved in melanin synthesis in vivo.

[0033] More specifically, tyrosinase activity refers to the activity of converting tyrosine residues in a polypeptide to DOPA (3,4-dihydroxyphenylalanine) residues and / or converting DOPA residues to DOPAquinone residues. The two-step reaction scheme in which tyrosine residues in a polypeptide are converted to DOPA residues and then converted to DOPAquinone residues is shown below. [ka]

[0034] As can be seen from the above reaction scheme, the reaction in which tyrosine residues are converted to DOPA residues and the reaction in which DOPA residues are converted to DOPAquinone residues are both oxidation reactions. Generally, tyrosinase can catalyze both of these oxidation reactions.

[0035] As mentioned above, tyrosinase is a protein widely conserved among various organisms, and its amino acid sequence and the like are publicly known. Specific examples of tyrosinases include tyrosinases derived from the genus Streptomyces, tyrosinases derived from actinomycetes other than Streptomyces, and tyrosinases derived from mushrooms. The amino acid sequence having tyrosinase activity may be the amino acid sequence of a known tyrosinase itself, or may be an amino acid sequence in which one or more amino acid residues have been substituted, deleted, or added from the amino acid sequence of a known tyrosinase, provided that the tyrosinase activity is not lost. In the present disclosure, the amino acid sequence having tyrosinase activity is preferably the amino acid sequence of a tyrosinase derived from the genus Streptomyces.

[0036] An example of an amino acid sequence having tyrosinase activity (SEQ ID NO: 2) is shown in Figure 12. The amino acid sequence represented by SEQ ID NO: 2 corresponds to tyrosinase derived from the genus Streptomyces. In the present disclosure, the amino acid sequence having tyrosinase activity may be used singly or in combination of two or more.

[0037] Amino acid sequences having tyrosinase activity in the present disclosure include the amino acid sequence set forth in SEQ ID NO: 2 and amino acid sequences having 70% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. The sequence identity can be 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0038] Whether a protein has tyrosinase activity can be evaluated by conventional methods. For example, it can be confirmed using a commercially available tyrosinase inhibitor screening kit. Alternatively, as shown in AJ Winder et al., "New assays for tyrosine hydroxylase and dopa," Eur J. Biochcm. f98, 317-326 (1991)), tyrosinase activity can be confirmed by reacting DOPAquinone, which is generated as the tyrosinase reaction proceeds, with 3-methyl-2-benzothiazolinonehydrazone (MBTH) and measuring the absorbance at 505 nm. Furthermore, tyrosinase activity can be confirmed according to the procedure in Test Example 2 described below, or can be predicted by computer simulation based on the amino acid sequence of the protein.

[0039] 1-4. Structure of the fusion tyrosinase of the present disclosure The fusion tyrosinase of the present disclosure has a structure in which the above-mentioned sequences (A) to (C) are linked in the order of (A)-(B)-(C). The sequences (A) to (C) can be linked directly to each other, or can be linked indirectly via an amino acid sequence other than the sequences (A) to (C). Furthermore, an amino acid sequence other than the sequences (A) to (C) can be included on the N-terminal and / or C-terminal side of the structure in which the sequences are linked in the order of (A)-(B)-(C).

[0040] The fusion tyrosinase of the present disclosure can also be bound to or fused with other peptides, oligopeptides, or proteins, such as albumin (e.g., serum albumin), protein tags (e.g., biotin, histidine tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag), fluorescent proteins (e.g., GFP, BFP, CFP, YFP, RFP), luminescent proteins (e.g., luciferase), secretory signal sequences (e.g., Igκ signal sequence), protease recognition sequences (e.g., TEV protease recognition sequence), expression-enhancing sequences, solubilization sequences, and multimerization domains.

[0041] Although not particularly limited, it is preferable that the fusion tyrosinase of the present disclosure has sequence (A) as sequence (a1): an amino acid sequence represented by SEQ ID NO: 1, or sequence (a2): an amino acid sequence having 70% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 1; and sequence (C) as sequence (c1): an amino acid sequence represented by SEQ ID NO: 2, or sequence (c2): an amino acid sequence having 70% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 2.

[0042] In the fusion tyrosinase of the present disclosure, it is more preferable that the sequence (A) is sequence (a1): an amino acid sequence represented by SEQ ID NO: 1, or sequence (a2): an amino acid sequence having 80% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 1; and the sequence (C) is sequence (c1): an amino acid sequence represented by SEQ ID NO: 2, or sequence (c2): an amino acid sequence having 80% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 2.

[0043] It is further preferred that the fusion tyrosinase of the present disclosure has sequence (A) as sequence (a1): an amino acid sequence represented by SEQ ID NO: 1, or sequence (a2): an amino acid sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 1; and sequence (C) as sequence (c1): an amino acid sequence represented by SEQ ID NO: 2, or sequence (c2): an amino acid sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 2.

[0044] It is particularly preferred that the fusion tyrosinase of the present disclosure has sequence (A) as sequence (a1): an amino acid sequence represented by SEQ ID NO: 1, or sequence (a2): an amino acid sequence having 95% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 1; and sequence (C) as sequence (c1): an amino acid sequence represented by SEQ ID NO: 2, or sequence (c2): an amino acid sequence having 95% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 2.

[0045] Furthermore, although not particularly limited, the fusion tyrosinase of the present disclosure preferably comprises sequence (d1): an amino acid sequence represented by SEQ ID NO: 5, or sequence (d2): an amino acid sequence with 70% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 5; more preferably comprises sequence (d1): an amino acid sequence represented by SEQ ID NO: 5, or sequence (d2): an amino acid sequence with 80% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 5; even more preferably comprises sequence (d1): an amino acid sequence represented by SEQ ID NO: 5, or sequence (d2): an amino acid sequence with 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 5; and particularly preferably comprises sequence (d1): an amino acid sequence represented by SEQ ID NO: 5, or sequence (d2): an amino acid sequence with 95% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 5.

[0046] Furthermore, although not particularly limited, the fusion tyrosinase of the present disclosure preferably consists of sequence (d1): the amino acid sequence represented by SEQ ID NO: 5, or sequence (d2): an amino acid sequence with 70% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 5; more preferably sequence (d1): the amino acid sequence represented by SEQ ID NO: 5, or sequence (d2): an amino acid sequence with 80% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 5; even more preferably sequence (d1): the amino acid sequence represented by SEQ ID NO: 5, or sequence (d2): an amino acid sequence with 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 5; and particularly preferably sequence (d1): the amino acid sequence represented by SEQ ID NO: 5, or sequence (d2): an amino acid sequence with 95% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 5.

[0047] The fusion tyrosinase of the present disclosure can also be chemically modified as long as the desired effect can be obtained.

[0048] The fusion tyrosinase of the present disclosure has a C-terminus containing a carboxy group (-COOH), a carboxylate (-COO - ), an amide group (-CONH2), or an ester group (-COOQ). Here, Q in the ester group can be any of C groups such as methyl, ethyl, n-propyl, isopropyl, and n-butyl. 1-6 Alkyl groups: cyclopentyl, cyclohexyl, etc. 3-8 Cycloalkyl groups such as phenyl and α-naphthyl 6-12 Aryl groups; phenyl-C such as benzyl and phenethyl 1-2 Examples thereof include an alkyl group and a pivaloyloxymethyl group.

[0049] In the fusion tyrosinase of the present disclosure, a carboxy group (or carboxylate) other than that at the C-terminus may be amidated or esterified.

[0050] In the fusion tyrosinase of the present disclosure, the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., a C group such as a formyl group or an acetyl group). 1-6Alkanoyl etc. C 1-6 those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.); 1-6 C such as alkanoyl group 1-6 Also included are those protected by an acyl group or the like, or conjugated proteins such as so-called glycoproteins to which sugar chains are bound.

[0051] 1-5. Method for producing the fusion tyrosinase of the present disclosure The fusion tyrosinase of the present disclosure can be produced by methods known in the art or methods that can be easily derived by a person skilled in the art from methods known in the art. For example, it can be produced by a method comprising the steps of culturing a host transformed with a polynucleotide containing a coding sequence for the fusion tyrosinase of the present disclosure and recovering a fraction containing the fusion tyrosinase of the present disclosure. Hereinafter, the polynucleotide may be referred to as the "polynucleotide of the present disclosure."

[0052] Specific embodiments of the polynucleotide of the present disclosure are not particularly limited, as long as they contain the coding sequence for the fusion tyrosinase of the present disclosure. Preferably, the polynucleotide of the present disclosure contains the coding sequence in a state capable of expressing the fusion tyrosinase of the present disclosure. The polynucleotide of the present disclosure may also contain other sequences in addition to the coding sequence. Examples of such other sequences include a secretory signal peptide coding sequence, a promoter sequence, an enhancer sequence, a repressor sequence, an insulator sequence, a replication origin, and a drug resistance gene coding sequence located adjacent to the coding sequence. Furthermore, the polynucleotide of the present disclosure may be a linear polynucleotide or a circular polynucleotide (e.g., a vector).

[0053] Specific examples of polynucleotides of the present disclosure include polynucleotides comprising the base sequence set forth in SEQ ID NO: 13, or a base sequence with 70% or more sequence identity to the base sequence set forth in SEQ ID NO: 13, and the base sequence set forth in SEQ ID NO: 14, or a base sequence with 70% or more sequence identity to the base sequence set forth in SEQ ID NO: 14, as well as polynucleotides comprising the base sequence set forth in SEQ ID NO: 17, or a base sequence with 70% or more sequence identity to the base sequence set forth in SEQ ID NO: 17. The sequence identity can be 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0054] In the present disclosure, it is preferable that a base sequence X' that does not have 100% sequence identity to a certain base sequence X does not cause a frameshift compared to the base sequence X. When the amino acid sequence encoded by the base sequence X is referred to as P and the amino acid sequence encoded by the base sequence X' is referred to as P', it is particularly preferable that P' is identical to P.

[0055] Polynucleotides such as DNA and RNA may be chemically modified, as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide can be substituted with chemically modified phosphate groups such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide can also be substituted with -OR (where R represents, for example, -CH, -CHCHOCH, -CHCHNHC(NH)NH, -CHCONHCH, or -CHCHCN). Furthermore, the base moiety (pyrimidine or purine) can also be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Modifications of the phosphate moiety or hydroxy moiety with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group are also possible, but are not limited to these.

[0056] The host to be transformed with the polynucleotide of the present disclosure is not particularly limited and may be, for example, Escherichia coli, eukaryotic cells, insect cells, mammalian cells, etc. From the viewpoint of protein expression efficiency, the host is preferably Escherichia coli.

[0057] The methods for transformation, culture, recovery, etc. are not particularly limited, and any method known in the art or a method that can be easily derived from a method known in the art can be employed.

[0058] The recovered fusion tyrosinase can be purified as needed. Purification can be carried out by methods known in the art or methods easily derived from methods known in the art. Such methods include, for example, chromatography, dialysis, etc.

[0059] Whether or not the desired polypeptide has been obtained can be confirmed by methods known in the art or methods that can be easily derived from methods known in the art. For example, after SDS-PAGE, proteins are visualized by CBB staining or the like, and whether or not the desired polypeptide has been obtained can be confirmed by whether or not a band with a molecular weight similar to that of the desired protein is observed. Alternatively, whether or not the desired protein has been obtained can be confirmed by Western blotting using an appropriate antibody after SDS-PAGE.

[0060] 1-6. Immobilization of fused tyrosinase The fusion tyrosinase of the present disclosure can be immobilized on any carrier as long as the effects of the present disclosure can be obtained. Examples of carriers include magnetic beads. When the fusion tyrosinase of the present disclosure is immobilized on magnetic beads, the fusion tyrosinase can be easily and efficiently removed from the reaction system.

[0061] 2. Uses of the Compositions of the Present Disclosure A composition of the present disclosure containing a fusion tyrosinase of the present disclosure is used to contact the fusion tyrosinase of the present disclosure with a target polypeptide containing a tyrosine residue. Preferably, the composition of the present disclosure is used to produce a modified polypeptide through said contact, wherein the modified polypeptide is a modified polypeptide containing a structure in which a DOPA residue and / or a DOPA quinone residue formed by conversion of the tyrosine residue is bound to an organic substance having a reactive functional group, and the reactive functional group is a functional group reactive with the DOPA residue and / or the DOPA quinone residue. Each element is described in detail below.

[0062] 2-1. Target polypeptide In the present disclosure, the term "polypeptide of interest" refers to a polypeptide to be contacted with the fusion tyrosinase of the present disclosure. The polypeptide of interest contains at least one tyrosine residue.

[0063] In the polypeptide structure, tyrosine residues near the N-terminus and / or C-terminus are predicted to be more easily accessible to the fusion tyrosinase of the present disclosure than tyrosine residues located further from the termini. Therefore, tyrosine residues near the N-terminus and / or C-terminus are more likely to be converted to DOPA residues and / or DOPA quinone residues than tyrosine residues located further from the termini. From this perspective, it is preferred that the subject polypeptides of the present disclosure contain tyrosine residues near the N-terminus and / or C-terminus.

[0064] Furthermore, as described below, the technology of the present disclosure can be used to convert tyrosine residues in a target polypeptide to DOPA residues and / or DOPAquinone residues, and then bind an organic substance to the DOPA residues and / or DOPAquinone residues via a reactive functional group. It is predicted that binding of the organic substance near the end of the polypeptide will have less of an effect on the three-dimensional structure and function of the polypeptide. From this perspective, it is preferable that the target polypeptide in the technology of the present disclosure contains a tyrosine residue near the N-terminus and / or C-terminus.

[0065] Although not particularly limited, the target polypeptide preferably contains at least one tyrosine residue within 20 amino acid residues from the N-terminus and / or within 20 amino acid residues from the C-terminus; more preferably within 15 amino acid residues from the N-terminus and / or within 15 amino acid residues from the C-terminus; even more preferably within 10 amino acid residues from the N-terminus and / or within 10 amino acid residues from the C-terminus; particularly preferably within 5 amino acid residues from the N-terminus and / or within 5 amino acid residues from the C-terminus; and most preferably has a tyrosine residue at the N-terminus and / or C-terminus. The upper limit of the above range may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues.

[0066] Furthermore, although not particularly limited, the target polypeptide may have (GSHY) at the N-terminus and / or C-terminus.n (n is a natural number). The range of the natural number n is not particularly limited, and may be, for example, 1 to 20. The upper or lower limit of the range may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The range of the natural number n is preferably 1 to 10, more preferably 1 to 8, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0067] The subject polypeptide may also be chemically modified. The description of chemical modifications described in the section "1-4. Structure of the fusion tyrosinase of the present disclosure" is also applicable to the subject polypeptide.

[0068] Furthermore, sortase A (EC: 3.4.22.70), a type of transpeptidase, can also be used to chemically modify a target polypeptide. Sortase A recognizes the LPXTG motif (SEQ ID NO: 9) and catalyzes the transfer of an amino group between a threonine (T) residue and a glycine (G) residue in a calcium-dependent manner. Therefore, any polypeptide having an LPXTG motif added thereto can be modified with any compound having a glycine residue added thereto by mixing the mixture and treating it with sortase A. Furthermore, it is believed that any compound having a terminal amino group and a catechol structure can be transferred to any polypeptide having an LPXTG motif added thereto by using sortase A.

[0069] Although not particularly limited, it is particularly preferred that the target polypeptide is an antibody or an antibody fragment having antigen-binding ability. In the present disclosure, the term "antibody" is used to encompass both monoclonal and polyclonal antibodies. The antibody fragment is not particularly limited as long as it contains heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3, and may be, for example, Fab, F(ab')2, minibody, scFv-Fc, Fv, scFv, diabody, triabody, tetrabody, etc.

[0070] 2-2. Contact between fusion tyrosinase and target polypeptide Tyrosine residues contained in a target polypeptide can be converted to DOPA residues and / or DOPAquinone residues through contact with the fusion tyrosinase of the present disclosure. The two-step reaction scheme in which a tyrosine residue in a polypeptide is converted to a DOPA residue and then the DOPA residue is further converted to a DOPAquinone residue is shown below. The amino acid residues shown in the scheme below are, from left to right, a tyrosine residue, a DOPA residue, and a DOPAquinone residue. [ka]

[0071] The specific manner of contacting the fusion tyrosinase of the present disclosure with the target polypeptide is not particularly limited. For example, the fusion tyrosinase of the present disclosure and the target polypeptide can be contacted (reacted) by mixing a composition of the present disclosure containing the fusion tyrosinase of the present disclosure with the target polypeptide. In this case, the reaction temperature, reaction time, composition of the reaction system, etc. are not particularly limited as long as the effects of the present disclosure are achieved.

[0072] For example, the reaction temperature can be 0 to 45°C, preferably 2 to 40°C, more preferably 2 to 37°C, further preferably 2 to 20°C, and particularly preferably 2 to 10°C. The reaction time can be, for example, about 10 minutes to 48 hours, about 30 minutes to 36 hours, or about 1 hour to 24 hours. The reaction time can be adjusted appropriately depending on other conditions such as the reaction temperature.

[0073] The composition of the reaction system is not particularly limited as long as the effects of the present disclosure can be obtained. It is particularly preferable that the reaction system is substantially free of proteases. In the present disclosure, "substantially free of proteases" means that the reaction system does not contain intentionally added proteases. In other words, "substantially free of proteases" does not exclude situations in which the reaction system inevitably contains trace amounts of proteases. Specific examples of proteases that may be inevitably present in the reaction system include proteases derived from cells that are brought into the reaction system when the fusion tyrosinase and / or target polypeptide is obtained by intracellular translation.

[0074] The reaction system may further contain other components as long as the effects of the present disclosure are obtained. Examples of other components include solvents, buffers, pH adjusters, chelating agents, etc.

[0075] During contact (reaction) between the fusion tyrosinase of the present disclosure and the target polypeptide, oxygen (O2) can be actively supplied to the reaction system. Specifically, for example, a gas containing oxygen (O2) can be blown into the liquid reaction system. As described above, both of the two-step reactions catalyzed by tyrosinase activity are oxidation reactions. Therefore, the presence of abundant oxygen in the reaction system can improve the efficiency of the oxidation reaction.

[0076] Furthermore, copper ions can be added to the reaction system during contact (reaction) between the fusion tyrosinase of the present disclosure and the target polypeptide. As described above, tyrosinase is a type III copper protein, and the presence of copper ions is required for tyrosinase activity. Therefore, the presence of abundant copper ions in the reaction system can improve the efficiency of the oxidation reaction that proceeds through tyrosinase activity. Note that when the fusion tyrosinase of the present disclosure is obtained by intracellular translation, copper ions derived from the cells are bound to the obtained fusion tyrosinase. Therefore, tyrosinase activity can be exerted even if copper ions are not added to the reaction system.

[0077] It is particularly preferable that the reaction system contains an organic substance having a reactive functional group, which will be described later. When the reaction system contains an organic substance having a reactive functional group, the modified polypeptide, which will be described later, can be efficiently obtained.

[0078] 2-3. Organic substances with reactive functional groups In the present disclosure, the term "reactive functional group" refers to a functional group reactive with a DOPA residue and / or a DOPA quinone residue. Functional groups reactive with a DOPA residue and / or a DOPA quinone residue are conventionally known.

[0079] The reactive functional group is preferably capable of reacting with a DOPA residue and / or a DOPA quinone residue under mild conditions (temperature, pH, etc.). Here, mild conditions are, for example, conditions under which the fusion tyrosinase of the present disclosure is not inactivated, more preferably conditions under which the fusion tyrosinase of the present disclosure is not inactivated and the target polypeptide is not denatured. Furthermore, it is particularly preferred that the reactive functional group is capable of reacting with a DOPA residue and / or a DOPA quinone residue under mild conditions and without the need for a metal catalyst or the like.

[0080] Specific examples of the reactive functional group include an amino group, a thiol group, an imidazole group, an alkynyl group, and a group represented by the following general formula (I): [ka] and the like.

[0081] Examples of organic substances having a group represented by the general formula (I) include those represented by the following general formula (III): [ka] an organic substance having a moiety represented by the following general formula (IV): [ka] and an organic substance having a moiety represented by the following general formula (V): [ka] Examples of the organic substance include an organic substance having a moiety represented by the following formula:

[0082] Further specific examples of organic substances having reactive functional groups are shown below. [ka] [ka] [ka]

[0083] In the present disclosure, the structure of the portion of an organic substance having a reactive functional group other than the reactive functional group is not particularly limited. The organic substance preferably has a portion capable of exerting physiological activity and / or pharmacological activity. More specifically, the organic substance having a reactive functional group preferably has a structure in which an arbitrary drug is linked to the reactive functional group. In this case, the drug may be linked directly to the reactive functional group, or may be linked via a linker and / or an arbitrary structure.

[0084] Furthermore, the organic substance having a reactive functional group can have a functional group reactive with an amino group, a thiol group, or the like, in addition to a functional group reactive with a DOPA residue and / or a DOPAquinone residue.

[0085] The molecular weight of the organic substance having a reactive functional group is not particularly limited as long as the effects of the present disclosure are achieved. For example, it can be 100 to 2,000, and preferably 150 to 1,200.

[0086] Examples of the linker include an alkyl linker, a polyoxyalkylene linker such as a PEG linker, a peptide linker, a disulfide linker, a thioether linker, an ester linker, a hydrazone linker, etc. The linker may also have a structure that can be cleaved by an enzyme present in a living body.

[0087] Examples of drugs include drugs with antitumor activity, drugs with cytotoxic activity, drugs with immunosuppressive activity, drugs with antibacterial activity, and drugs for bioimaging.

[0088] 2-4. Modified Polypeptides The modified polypeptide produced by contacting the fusion tyrosinase with the target polypeptide preferably comprises a structure in which the DOPA residue and / or DOPA quinone residue, which are formed by converting the tyrosine residue, are bound to an organic substance having a reactive functional group. For example, when the DOPA quinone residue is bound to an organic substance having a moiety represented by the general formula (III), the following general formula (VI): [ka] The resulting modified polypeptide has the structure:

[0089] The modified polypeptide obtained by the technology of the present disclosure is preferably a bioconjugate, and more preferably an antibody-drug conjugate (ADC). In this disclosure, the term "bioconjugate" refers to a biopharmaceutical having a structure in which an arbitrary compound is directly or indirectly bound to a polypeptide. When the polypeptide is an antibody or an antigen-binding fragment of an antibody and the arbitrary compound is a drug, the bioconjugate is specifically referred to as a "drug-antibody conjugate." Hereinafter, when the term "antibody" is used in the context of a drug-antibody conjugate, it is intended to encompass both an antibody and an antigen-binding fragment of an antibody.

[0090] An example of an ADC is a conjugate consisting of an antibody with high specificity for cancer cells and a drug (payload) with antitumor activity. The mechanism of the ADC's anticancer action is as follows: the ADC leaks from neovasculature due to increased vascular permeability through the enhanced permeability and retention (EPR) effect, and accumulates around the cancer tissue; the antibody moiety of the ADC binds to antigens on the surface of cancer cells, leading to intracellular uptake; and the drug is cleaved from the ADC by intracellular enzymes, resulting in antitumor activity. As can be seen from these mechanisms, ADCs have excellent functionality as drug delivery systems (DDSs) by selectively delivering drugs to target tissues and cells. This allows for reduced side effects and lower drug dosages compared to systemic administration of drugs without ADCs.

[0091] 3. Method for Producing Modified Polypeptides The present disclosure also encompasses a method for producing a modified polypeptide, the method comprising the step of contacting a copper transport protein fusion tyrosinase with a polypeptide of interest to convert at least one tyrosine residue contained in the polypeptide of interest into a DOPA residue and / or a DOPAquinone residue, wherein the copper transport protein fusion tyrosinase comprises sequence (A): an amino acid sequence having copper transport protein function, sequence (B): a linker sequence, and sequence (C): an amino acid sequence having tyrosinase activity, and the sequences (A) to (C) are linked in the order (A)-(B)-(C). This production method encompassed by the present disclosure may be referred to as the "production method of the present disclosure." The matters described in Sections "1. Fusion tyrosinase of the present disclosure" to "2. Uses of the composition of the present disclosure" are incorporated by reference into the production method of the present disclosure.

[0092] In the technology of the present disclosure, modified polypeptides are produced, for example, by the following process: contacting a target polypeptide with the fusion tyrosinase of the present disclosure to convert at least one tyrosine residue in the polypeptide into a DOPA residue and / or a DOPA quinone residue; and binding an organic substance having a reactive functional group to the DOPA residue and / or DOPA quinone residue in the polypeptide via the reactive functional group.

[0093] Furthermore, when the modified polypeptide obtained by the technology of the present disclosure is an ADC, the modified polypeptide is produced, for example, by the following process: contacting an antibody (target polypeptide) with the fusion tyrosinase of the present disclosure to convert at least one tyrosine residue in the antibody into a DOPA residue and / or a DOPAquinone residue; and binding an organic substance having a reactive functional group and a moiety having pharmacological activity to the DOPA residue and / or DOPAquinone residue in the antibody via the reactive functional group.

[0094] As described above, the method for producing a modified polypeptide using the technology of the present disclosure is understood to include two steps: (i) converting tyrosine residues in a target polypeptide to DOPA residues and / or DOPAquinone residues, and (ii) binding an organic substance having a reactive functional group to the DOPA residue and / or DOPAquinone residue in the polypeptide via the reactive functional group. These two steps can be performed independently (i.e., step (ii) after step (i) is completed) or in parallel (i.e., step (i) and step (ii) are performed almost simultaneously).

[0095] When the two steps are carried out independently, for example, after a step of converting a tyrosine residue in a target polypeptide into a DOPA residue and / or a DOPAquinone residue in a reaction system containing a target polypeptide and a fusion tyrosinase of the present disclosure but not containing an organic substance having a reactive functional group, an organic substance having a reactive functional group can be added to the reaction system to bind the organic substance to the DOPA residue and / or DOPAquinone residue in the polypeptide via the reactive functional group.

[0096] When the two steps are carried out in parallel, for example, by preparing a reaction system containing a target polypeptide, the fusion tyrosinase of the present disclosure, and an organic substance having a reactive functional group, the tyrosine residue in the target polypeptide is converted into a DOPA residue and / or a DOPA quinone residue, and at almost the same time, the organic substance having a reactive functional group can be bound to the DOPA residue and / or the DOPA quinone residue.

[0097] As mentioned above, DOPA residues and DOPAquinone residues are known to contribute to the formation of intermolecular crosslinks in polypeptides. When a polypeptide forms a complex through intermolecular crosslinks via DOPA residues and / or DOPAquinone residues, it is predicted that the reactivity with organic substances having reactive functional groups will decrease due to steric hindrance, etc. Therefore, from the perspective of efficiently producing modified polypeptides, it is preferable to perform the above two steps in parallel. When the above two steps are performed in parallel, the DOPA residues and / or DOPAquinone residues converted from tyrosine residues are expected to bind to organic substances having reactive functional groups before forming intermolecular crosslinks.

[0098] 4. Possible mechanism The fusion tyrosinase of the present disclosure is predicted to have a structure in which at least a portion of the portion having tyrosinase activity is shielded by the portion having copper transport protein function. This structure is thought to partially suppress tyrosinase activity. Therefore, the technology of the present disclosure is thought to be able to efficiently produce organic substance-modified polypeptides while suppressing the formation of complexes between polypeptides (intermolecular cross-linking) due to the adhesive properties of DOPA residues and DOPA quinone residues.

[0099] Furthermore, as described above, the fusion tyrosinase of the present disclosure has a structure in which at least a portion of the portion having tyrosinase activity is shielded by the portion having copper transport protein function, and therefore it is believed that tyrosine residues near the terminus of the polypeptide, which have less steric hindrance, are preferentially converted to DOPA residues and / or DOPAquinone residues. Generally, when modifying a polypeptide with an organic substance, it is predicted that modification near the terminus of the polypeptide is less likely to affect the three-dimensional structure and function of the polypeptide. Therefore, the technology of the present disclosure, which can preferentially convert tyrosine residues near the terminus to DOPA residues and / or DOPAquinone residues, can be said to be useful even when it is desired to bind an organic substance to the polypeptide while maintaining its function.

[0100] In this specification, the term "comprising" includes "essentially consisting of" and "consisting of" in addition to "containing." Furthermore, the present disclosure encompasses all arbitrary combinations of the constituent elements described in this specification.

[0101] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0102] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0103] Test Example 1. Preparation of copper transport protein fused tyrosinase 1-1. Construction of a copper transport protein-fused tyrosinase expression plasmid A copper transport protein-fused tyrosinase expression plasmid was constructed using the known pET system. A model diagram of the plasmid is shown in Figure 1. In Figure 1, "orf438" represents the nucleotide sequence (SEQ ID NO: 13) encoding the copper transport protein (SEQ ID NO: 1). "GS linker" represents the nucleotide sequence (SEQ ID NO: 15) encoding a linker (SEQ ID NO: 3) consisting of the amino acid sequence GS(G4S)3SG. "Cleavage sequence" represents the nucleotide sequence (SEQ ID NO: 15) encoding a sequence-specific protease recognition sequence (SEQ ID NO: 4). "melC" represents the nucleotide sequence (SEQ ID NO: 14) encoding tyrosinase (SEQ ID NO: 2). "His-tag" represents the nucleotide sequence encoding a tag consisting of six histidines. Note that orf438 and melC are derived from Streptomyces bacteria. The full-length amino acid sequence of the copper transport protein-fused tyrosinase expressed by the plasmid is shown in Figure 12 (SEQ ID NO: 5).

[0104] 1-2. Transformation of E. coli (HMS174(DE3)) 2 μL of the plasmid solution prepared in 1-1 was added to 200 μL of competent cells of E. coli HMS174(DE3) and allowed to stand on ice for 5 minutes. Then, the entire E. coli solution was spread with a Conlarge stick onto LB agar medium containing an antibiotic (ampicillin, final concentration 75 μg / mL) and cultured overnight at 37°C.

[0105] 1-3. Protein production The E. coli colonies obtained in 1-2 were inoculated into 2 mL of ampicillin-containing LB liquid medium, and then culture was initiated at 37°C. During the culture, the culture was scaled up to 100 mL of ampicillin-containing LB liquid medium, and the turbidity (OD 600 Cultivation was continued at 37°C until the turbidity exceeded 0.6. After confirming that the turbidity exceeded 0.6, isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 0.15 mM, and copper sulfate was further added to a final concentration of 50 μM, followed by overnight cultivation at 25°C. After cultivation, the entire culture medium was centrifuged at 6000 rpm at 4°C for 10 minutes to collect the bacterial cells. The collected bacterial cells were stored at -30°C until further testing.

[0106] 1-4. Confirmation of protein expression One mL of bacterial cells from the culture obtained in 1-3 was added to a 1.5 mL plastic tube and suspended in 50 μL of 1× PBS. The suspended bacterial cells were sonicated using an ultrasonicator (disruption: 20 seconds, rest: 40 seconds, 10 cycles) and then centrifuged at 15,000 rpm at 4°C for 5 minutes. After centrifugation, the soluble fraction (supernatant) and insoluble fraction (precipitate) were separated. 8 μL of the soluble fraction was subjected to SDS-PAGE, as described below. The insoluble fraction was suspended in 50 μL of 1× SDS Sample buffer, and 4 μL of this suspension was subjected to SDS-PAGE, as described below.

[0107] The composition of the electrophoresis gel used for SDS-PAGE is shown in the table below. The electrophoresis gel was prepared by layering a concentration gel on top of a separation gel. The acrylamide concentration in the separation gel was 10 w / v%. In the table, WIDE RANGE Gel Preparation Buffer (4x) for PAGE refers to WIDE RANGE Gel Preparation Buffer (4x) for PAGE (Nacalai Tesque). APS refers to ammonium persulfate, and its concentration "40%" refers to 40 w / v%. TEMED refers to N,N,N',N'-tetramethylethylenediamine. dH2O refers to distilled water.

[0108] [Table 1]

[0109] [Table 2]

[0110] An appropriate amount of 5xSDS sample buffer was added to each sample subjected to electrophoresis, followed by denaturation at 95°C for 2 minutes. After denaturation, each sample was applied to the electrophoresis gel. Electrophoresis was performed at a constant current. All SDS-PAGE described in this example was performed using the method described above.

[0111] After electrophoresis, the gel was stained with CBB to visualize the proteins. The results are shown in Figure 2. From left to right, the lanes are molecular weight markers, soluble fraction, and insoluble fraction. Based on the sequence of the copper transport protein-fused tyrosinase expression plasmid prepared in 1-1, the molecular weight of the target fusion protein (SEQ ID NO: 5) is predicted to be 45,967. As shown in Figure 2, a band was observed between the molecular weight markers of 40,000 and 50,000 in both the soluble and insoluble fractions. A particularly prominent band was observed in the soluble fraction. Therefore, the present inventors used the soluble fraction to purify the protein, as described below.

[0112] 1-5. Protein purification 100 mL of E. coli culture medium obtained in 1-3 was added to 100 mL of bacterial cells in 1x PBS and suspended. The suspended bacterial cells were sonicated using an ultrasonicator (disruption: 20 seconds, rest: 40 seconds, 7 cycles). The suspension after sonication was almost transparent. The suspension after sonication was centrifuged at 6000 rpm at 4°C for 10 minutes, and the supernatant was used as a bacterial cell extract (hereinafter, this bacterial cell extract may be referred to as "pre-column").

[0113] After adding 800 μL of Ni-NTA agarose gel resin suspension to a plastic column, 2 mL of 1×PBS was added twice to equilibrate the column. The bacterial cell extract was applied to the equilibrated column. After application, the liquid coming out from the bottom of the column was collected (this liquid may be referred to as the "flow-through fraction").

[0114] After collecting the flow-through fraction, 2 mL of wash buffer was applied to the column and the liquid that came out from the bottom of the column was collected (this liquid is sometimes referred to as "wash fraction 1"). This procedure of applying 2 mL of wash buffer and then collecting the liquid that came out from the bottom of the column was repeated three times in total to obtain wash fractions 1 to 3. The composition of the wash buffer is shown in the table below. In the table, % indicates v / v%.

[0115] [Table 3]

[0116] After washing three times as described above, 1 mL of Elution buffer was applied to the column and the liquid that came out from the bottom of the column was collected (this liquid is sometimes referred to as "elution fraction 1"). Similarly, 1 mL of Elution buffer was applied and the liquid that came out from the bottom of the column was collected three times, yielding elution fractions 1 to 3. The composition of the Elution buffer is shown in the table below. In the table, % indicates v / v%.

[0117] [Table 4]

[0118] SDS-PAGE was performed on 4 μL of each fraction using the method described in 1-4. After electrophoresis, the gel was stained with CBB to visualize the proteins. The results are shown in Figure 3. From left to right, the lanes are molecular weight marker, pre-column, flow-through fraction, wash fractions 1 to 3, and elution fractions 1 to 3. As shown in Figure 3, a particularly intense band predicted to be copper transport protein fusion tyrosinase was observed in elution fraction 1. Elution fraction 1 was subjected to the subsequent concentration procedure.

[0119] Elution fraction 1 was added to the filter cup of an Amicon Ultra-15 10K (Merck) and centrifuged at 5000 × g and 4 ° C to concentrate to 500 μL. 3.5 mL of 1 × PBS was added to the filter cup and centrifuged at 5000 × g and 4 ° C to concentrate to 500 μL. 3.5 mL of 1 × PBS was added to the filter cup again and centrifuged at 5000 × g and 4 ° C to concentrate to 500 μL. 1.5 mL of 1 × PBS was then added to the filter cup and centrifuged at 5000 × g and 4 ° C to concentrate to 200 μL. This concentrate was used for the subsequent dialysis procedure.

[0120] The concentrated solution was added to the dialysis membrane of a Slide-A-Lyzer™ MINI Dialysis Device, 10K MWCO (Thermo Fisher Scientific). Dialysis buffer containing copper sulfate added to a final concentration of 50 μM was added to the tube of the Slide-A-Lyzer MINI Dialysis Device, and the dialysis membrane was set in place. The tube was stirred at 4°C overnight to exchange the buffer. The protein solution after buffer exchange was stored at -30°C until further testing. The composition of the dialysis buffer is shown in the table below. In the table below, % indicates v / v%.

[0121] [Table 5]

[0122] Test Example 2. Evaluation of copper transport protein fused tyrosinase 2-1. Evaluation of crosslinking reaction (reference example) In the copper transport protein fusion tyrosinase obtained in Test Example 1, the active site of the tyrosinase portion is shielded by the copper transport protein portion. In this example, this copper transport protein fusion tyrosinase may be referred to simply as "fusion tyrosinase." Because the linker portion of this fusion tyrosinase contains a protease recognition sequence, it is believed that by cleaving the fusion tyrosinase with a protease, the copper transport protein portion can be separated from the active site of the tyrosinase portion. The present inventors first evaluated the intermolecular crosslinking reaction using this fusion tyrosinase. Details are described below.

[0123] 2-1-1. Cross-linking reaction using a protein substrate with a tyrosine residue at its C-terminus Tyrosinase converts tyrosine residues in polypeptides to DOPA residues, and then converts the DOPA residues to DOPAquinone residues. That is, tyrosinase converts tyrosine residues in polypeptides to DOPAquinone residues through a two-step reaction. DOPA residues and DOPAquinone residues are known to contribute to intermolecular cross-linking. Therefore, the present inventors used proteins with a tyrosine residue at the C-terminus as substrates and evaluated the complex formation resulting from intermolecular cross-linking as an indicator of tyrosinase activity.

[0124] More specifically, a protein (SEQ ID NO: 10) in which a tyrosine residue was added to the C-terminus of LgBiT via a histidine tag was used as a substrate. LgBiT is an approximately 18 kDa NanoLuc™ luciferase fragment. This protein is sometimes referred to as "LgBiT-Y." LgBiT-Y was expressed and purified according to the method described in Test Example 1. The sequence of LgBiT-Y is shown in FIG. 12 (SEQ ID NO: 10). The three-dimensional structure of LgBiT-Y predicted by the protein structure prediction program AlphaFold2 (DeepMind) is shown in FIG. 4. In FIG. 4, tyrosine residues are shown in green. As shown in FIG. 4, in the three-dimensional structure of LgBiT-Y, the C-terminal tyrosine residue is predicted to be exposed on the protein surface.

[0125] To evaluate the activity of fusion tyrosinase, reaction mixtures were prepared with (+) or without (-) fusion tyrosinase, with (+) or without (-) 3C Protease, and with (+) or without (-) substrate (LgBiT-Y). The fusion tyrosinase content was 0.067 μg / μL, the 3C Protease content was 0.033 μg / μL, and the substrate (LgBiT-Y) content was 6.7 μM. 10% v / v 10x PBS was added to each reaction mixture, and the total volume was adjusted to 15 μL with distilled water.

[0126] Each reaction mixture was incubated at 4°C for 20 hours. After incubation, SDS-PAGE was performed as described in 1-4. After electrophoresis, the gel was stained with CBB to visualize the proteins. The results are shown in Figure 5. The molecular weight of the fusion tyrosinase is 45,967, that of the 3C protease is approximately 47,000, and that of the LgBiT-Y is 18,957. Furthermore, the molecular weights of the fragments generated when the fusion tyrosinase is cleaved with the 3C protease are 14,027 for the portion containing the copper transport protein (orf438) and 31,959 for the portion containing the tyrosinase (melC). As shown by the box in Figure 5, only in the rightmost lane containing the fusion tyrosinase, 3C protease, and substrate (LgBiT-Y), the 3C protease band with a molecular weight of approximately 47,000 disappeared, and multiple bands with molecular weights of over 50,000 were observed.

[0127] 2-1-2. Cross-linking reaction using proteins with C-terminal cysteine ​​residues as substrates A similar test to that in 2-1-1 was also performed using a protein in which a cysteine ​​residue was added to the C-terminus of LgBiT via a histidine tag (sometimes referred to as "LgBiT-C") as a substrate instead of LgBiT-Y. The results are shown in Figure 6. The molecular weight of LgBiT-C is 18,897.

[0128] As shown in Figure 6, unlike when LgBiT-Y was used as the substrate, when LgBiT-C was used, no bands with molecular weights of 50,000 or greater were observed, even in the rightmost lane containing all of the fusion tyrosinase, 3C Protease, and substrate (LgBiT-C) (the relevant area is boxed in the figure). Furthermore, a band representing 3C Protease with a molecular weight of approximately 47,000 was clearly observed in this lane.

[0129] 2-1-3. Summary This test example suggests that the inhibition of tyrosinase activity is relieved by the protease recognizing and cleaving the protease recognition sequence in the fusion tyrosinase. It also suggests that polypeptides having a terminal tyrosine residue are more likely to form complexes with tyrosinase activity.

[0130] 2-2. Evaluation of modification reactions The DOPA quinone residue may be an amino group, a thiol group, an imidazole group, an alkynyl group, or a quinone having the following general formula (I): [ka] The present inventors mixed and reacted the copper transport protein fusion tyrosinase obtained in Test Example 1, the LgBiT-Y having a tyrosine residue at the C-terminus used in Test 2-1, and an organic substance having a reactive functional group, and evaluated whether LgBiT-Y was modified by the organic substance. Details are described below.

[0131] 2-2-1. Modification with 5-FAM-PEG3-BCN-1 (4°C, 20 hours) 5-FAM-PEG3-BCN, also known as 5-carboxyfluorescein-PEG3-BCN, has a structure in which 5-carboxyfluorescein is bound to a BCN moiety via a PEG chain. The BCN moiety has a group represented by the general formula (I) above and is reactive with DOPA quinone residues. 5-carboxyfluorescein is a fluorescent molecule. The structural formula of 5-FAM-PEG3-BCN is shown below. In this example, 5-FAM-PEG3-BCN is sometimes referred to as "fluorescent BCN." [ka]

[0132] As described above, fluorescent BCN can bind to DOPAquinone residues via the BCN moiety. Therefore, if the tyrosine residue in LgBiT-Y is converted to a DOPAquinone residue by the fused tyrosinase, fluorescent BCN can bind to the DOPAquinone residue. LgBiT-Y modified with fluorescent BCN is considered to be fluorescently detectable. Therefore, the present inventors evaluated by fluorescence detection whether the following series of reactions occurred: (i) the tyrosine residue in LgBiT-Y is converted to a DOPAquinone residue by the fused tyrosinase; and (ii) the DOPAquinone residue is modified with fluorescent BCN.

[0133] More specifically, reaction mixtures were prepared with (+) or without (-) fusion tyrosinase, with (+) or without (-) 3C Protease, with (+) or without (-) substrate (LgBiT-Y), and with (+) or without (-) fluorescent BCN. Reaction mixtures were also prepared using LgBiT-C instead of LgBiT-Y. The fusion tyrosinase content was 0.067 μg / μL. The 3C Protease content was 0.033 μg / μL. The substrate (LgBiT-Y or LgBiT-C) content was 6.7 μM. The fluorescent BCN content was 100 μM. Each reaction mixture was supplemented with 10% 10x PBS and brought to a total volume of 15 μL with distilled water.

[0134] Each reaction mixture was incubated at 4°C for 20 hours. After incubation, SDS-PAGE was performed as described in 1-4. The electrophoretic gel was photographed using an imager (ChemDoc, Bio-Rad) equipped with a CCD camera (LAS3000, Fujifilm) to detect fluorescence from the 5-carboxyfluorescein moiety. The gel was also stained with CBB to visualize the proteins. The results of the CBB staining are shown in Figure 7 (left), and the results photographed with the CCD camera are shown in Figure 7 (right). The molecular weight of the fusion tyrosinase is 45,967, the molecular weight of the 3C protease is approximately 47,000, the molecular weight of LgBiT-Y is 18,957, and the molecular weight of LgBiT-C is 18,897. Furthermore, the molecular weight of each fragment generated when the fusion tyrosinase was cleaved with 3C protease was 14,027 for the portion containing the copper transport protein (orf438) and 31,959 for the portion containing tyrosinase (melC).

[0135] As shown on the left side of Figure 7, the substrate was LgBiT-Y, and only in the lane to which the sample containing fusion tyrosinase, substrate, and fluorescent BCN, but no protease, was applied, was a band shifted toward higher molecular weights, predicted to be LgBiT-Y (indicated by an arrow in the figure). Furthermore, as shown on the right side of Figure 7, a fluorescent signal was detected only from this band. These results suggest that the fusion tyrosinase produced a modified polypeptide in which fluorescent BCN was attached to LgBiT-Y.

[0136] On the other hand, when the substrate was LgBiT-Y and the mixture contained all of fusion tyrosinase, protease, substrate, and fluorescent BCN, and when the substrate was LgBiT-C, no band with a detectable fluorescent signal was observed.

[0137] These results suggest that fluorescent BCN-modified polypeptides can be efficiently obtained from fusion tyrosinase in which the tyrosinase active site is shielded by the copper transport protein portion. On the other hand, if the linker connecting the copper transport protein portion and the tyrosinase portion is cleaved with a protease to separate the copper transport protein portion from the tyrosinase portion (i.e., if the tyrosinase active site is exposed), it may not be possible to obtain fluorescent BCN-modified polypeptides.

[0138] 2-2-2. Modification with 5-FAM-PEG3-BCN-2 (37°C, 1 hour) Next, the inventors evaluated the modification of LgBiT-Y with fluorescent BCN in the same manner as in 2-2-1, except that each reaction solution was incubated at 37°C for 1 hour. Note that no protease-containing reaction solution was prepared in this test. The results are shown in Figure 8.

[0139] As in 2-2-1, the substrate in this experiment was LgBiT-Y. A band predicted to be LgBiT-Y, shifted toward higher molecular weight, was observed only in the lane containing the sample containing fusion tyrosinase, substrate, and fluorescent BCN (indicated by an arrow in the figure). Furthermore, as shown in the right side of Figure 8, a fluorescent signal was detected only from this band. These results suggest that the fusion tyrosinase produced a modified polypeptide in which fluorescent BCN was attached to LgBiT-Y.

[0140] However, in the CBB staining of the electrophoresis gel, the band predicted to be LgBiT-Y, which had shifted to the high molecular weight side, was fainter than that observed in 2-2-1. These results suggest that the amount of fluorescent BCN-modified polypeptide produced in this test, where the reaction mixture was reacted at 37°C for 1 hour, was smaller than that in 2-2-1, where the reaction mixture was reacted at 4°C for 20 hours.

[0141] 2-2-3. Modification with MBTH Next, the present inventors attempted to modify the polypeptide with an organic substance other than fluorescent BCN. Specifically, they attempted to modify the polypeptide with MBTH (3-Methyl-2-benzothiazolinonehydrazone) instead of the fluorescent BCN used in Test Examples 2-2-1 and 2-2-2.

[0142] MBTH is known to produce a red color when combined with DOPAquinone. The series of reactions in which DOPA is converted to DOPAquinone by tyrosinase and DOPAquinone reacts with MBTH to produce a red substance are shown below. [ka]

[0143] The present inventors hypothesized that DOPAquinone residues in polypeptides would react with MBTH to produce a red substance, similar to the reaction with free DOPAquinone, and conducted this study. Specifically, reaction mixtures were prepared with (+) or without (-) fusion tyrosinase, with (+) or without (-) substrate (LgBiT-Y), and with (+) or without (-) MBTH. Reaction mixtures were also prepared using LgBiT-C instead of LgBiT-Y. The fusion tyrosinase concentration was 0.067 μg / μL. The substrate (LgBiT-Y or LgBiT-C) concentration was 6.7 μM. The MBTH concentration was 134 μM. Each reaction mixture was supplemented with 10% 10x PBS (v / v), and the total volume was adjusted to 15 μL with distilled water.

[0144] Each reaction solution was incubated at 4°C for 20 hours. After the incubation, SDS-PAGE was performed using the method described in 1-4. No visible red bands were observed in the gel after electrophoresis (data not shown). The gel was stained with CBB to visualize the proteins. The results are shown in Figure 9.

[0145] As shown in Figure 9, the substrate was LgBiT-Y, and only in the lane to which the sample containing fusion tyrosinase, substrate, and MBTH was applied was a band shifted toward higher molecular weights, predicted to be LgBiT-Y (indicated by an arrow in the figure). This result suggests that the fusion tyrosinase produced a modified polypeptide in which MBTH was attached to LgBiT-Y.

[0146] 2-3. Study of magnetic bead-immobilized fusion tyrosinase Next, the present inventors attempted to carry out a polypeptide modification reaction using fusion tyrosinase immobilized (bound) to magnetic beads, thereby efficiently removing the fusion tyrosinase from the reaction system after the desired modified polypeptide was produced. The specific method is described below.

[0147] 2-3-1. Immobilization of fusion tyrosinase on magnetic beads 0.5 mg of magnetic beads ("Ts beads", Tamagawa Seiki Co., Ltd.) was centrifuged at 15,000 rpm at 4°C for 5 minutes, and the supernatant was removed. 100 μL of protein immobilization buffer was added, and the beads were dispersed using ultrasound. After dispersion, the beads were centrifuged at 15,000 rpm at 4°C for 5 minutes, and the supernatant was removed. 100 μL of protein immobilization buffer was added again, and the steps of dispersion in 100 μL of protein immobilization buffer, centrifugation, and supernatant removal were repeated three times in total. The composition of the protein immobilization buffer is shown in the table below.

[0148] [Table 6]

[0149] The fusion tyrosinase obtained in Test Example 1 was diluted to 10 μM with protein immobilization buffer. 100 μL of this diluted solution was added to the magnetic beads that had been washed as described above and the supernatant removed, and the solution was dispersed by ultrasonication and then stirred overnight at 4°C. After stirring, the solution was centrifuged at 15,000 rpm at 4°C for 5 minutes, and the supernatant was removed. 100 μL of protein immobilization buffer was added to the magnetic beads from which the supernatant had been removed, and the beads were dispersed by ultrasonication. After dispersion, the solution was centrifuged at 15,000 rpm at 4°C for 5 minutes, and the supernatant was removed.

[0150] After removing the supernatant as described above, 100 μL of 1 M Tris-HCl (pH 8.0) was added to the magnetic beads, which were dispersed by ultrasonication and then stirred overnight at 4°C. After stirring, the beads were centrifuged at 15,000 rpm at 4°C for 5 minutes, and the supernatant was removed. 100 μL of protein immobilization buffer was added to the magnetic beads, from which the supernatant was removed, and the beads were dispersed by ultrasonication. After dispersion, the beads were centrifuged at 15,000 rpm at 4°C for 5 minutes, and the supernatant was removed. 100 μL of protein immobilization buffer was added again, and the process of dispersion in 100 μL of protein immobilization buffer, centrifugation, and supernatant removal was repeated three times in the same manner.

[0151] After removing the final supernatant, 100 μL of protein immobilization buffer containing copper sulfate at a final concentration of 50 μM was added, and the beads were dispersed by ultrasonication. The beads were stored at 4°C until further experiments.

[0152] 2-3-2. Modification reaction using magnetic bead-immobilized fusion tyrosinase Reaction mixtures were prepared with (+) or without (-) fusion tyrosinase, with (+) or without (-) substrate (LgBiT-Y), and with (+) or without fluorescent BCN. Fusion tyrosinase was tested both without and with immobilization on magnetic beads. The content of the unimmobilized fusion tyrosinase was 0.067 μg / μL. The content of the immobilized fusion tyrosinase was 0.134 μg / μL (excluding the weight of magnetic beads). The content of the substrate (LgBiT-Y) was 6.7 μM. The content of fluorescent BCN was 100 μM. 10% 10x PBS was added to each reaction mixture, and the total volume was adjusted to 15 μL with distilled water.

[0153] Each reaction solution was incubated at 4°C for 20 hours. After the incubation, SDS-PAGE was performed using the method described in 1-4. For samples containing fusion tyrosinase immobilized on magnetic beads, the beads were removed using a magnet after the incubation and the resulting solution was subjected to SDS-PAGE. The gel after electrophoresis was photographed with an imager equipped with a CCD camera, and fluorescence derived from the 5-carboxyfluorescein moiety was detected. The gel was also stained with CBB to visualize the protein. The results of the CBB staining are shown on the left side of Figure 10, and the results photographed with the imager equipped with a CCD camera are shown on the right side of Figure 10.

[0154] As shown on the left side of Figure 10, the band representing the fusion tyrosinase disappeared in the immobilized lane, suggesting that the fusion tyrosinase was removed from the solution after the reaction along with the magnetic beads.

[0155] Furthermore, in the case of immobilization, as in the case of no immobilization, a band predicted to be LgBiT-Y shifted toward the high molecular weight side was observed in the lane to which a sample containing fusion tyrosinase, substrate, and fluorescent BCN was applied (indicated by an arrow in the figure). Furthermore, as shown on the right side of Figure 10, a fluorescent signal was also detected from this band. These results suggest that, similar to the case of using fusion tyrosinase without immobilization, when fusion tyrosinase was immobilized on magnetic beads, a modified polypeptide in which fluorescent BCN was attached to LgBiT-Y was produced.

[0156] These results suggest that by performing a polypeptide modification reaction using fusion tyrosinase immobilized on magnetic beads, the fusion tyrosinase can be efficiently removed from the reaction system after the desired modified polypeptide is produced.

[0157] 2-4. Examination of the terminal structure of the substrate polypeptide In all of the above experiments, LgBiT-Y, which has a tyrosine residue at the C-terminus, was used as the reaction substrate. Therefore, the present inventors investigated whether the modification reaction would occur similarly when polypeptides with other terminal structures were used as substrates.

[0158] Specifically, LgBiT-GSHYx1 (SEQ ID NO: 11) or LgBiT-GSHYx2 (SEQ ID NO: 12) was used as a substrate instead of LgBiT-Y. LgBiT-GSHYx1 has a structure in which one GSHY sequence is added to the C-terminus of LgBiT. LgBiT-GSHYx2 has a structure in which two GSHY sequences are added to the C-terminus of LgBiT. Furthermore, while LgBiT-Y has a histidine tag on the C-terminus, LgBiT-GSHYx1 and LgBiT-GSHYx2 have histidine tags on the N-terminus. LgBiT-GSHYx1 and LgBiT-GSHYx2 were prepared by constructing a protein expression vector, followed by expression and purification using the method described in Test Example 1.

[0159] LgBiT-GSHYx1 or LgBiT-GSHYx2 prepared as described above was reacted with fluorescent BCN. More specifically, for LgBiT-GSHYx1 and LgBiT-GSHYx2, reaction solutions were prepared with (+) or without (-) fusion tyrosinase, with (+) or without (-) fluorescent BCN, with (+) or without (-) copper ion addition, and with (+) or without (-) oxygen substitution. The fusion tyrosinase concentration was 0.067 μg / μL. The substrate (LgBiT-GSHYx1 or LgBiT-GSHYx2) concentration was 6.7 μM. The fluorescent BCN concentration was 100 μM. Copper ion addition was performed by adding 5 μM CuSO4. Oxygen substitution was performed by blowing oxygen into the reaction tube from an oxygen canister. In addition, 10x PBS was added to each reaction solution at 10 v / v%, and the total volume was adjusted to 15 μL with distilled water.

[0160] Each reaction mixture was incubated at 4°C for 20 hours. After incubation, SDS-PAGE was performed using the method described in 1-4. The gel after electrophoresis was photographed using an imager equipped with a CCD camera, and the fluorescence derived from the 5-carboxyfluorescein moiety was detected. The gel was also stained with CBB to visualize the proteins. The results of the CBB staining are shown on the left side of Figure 11, and the results photographed using the imager equipped with a CCD camera are shown on the right side of Figure 11.

[0161] First, in the lanes where samples containing fusion tyrosinase and fluorescent BCN were applied, a fluorescent band was detected, which was presumably due to the attachment of fluorescent BCN to the substrate (LgBiT-GSHYx1 or LgBiT-GSHYx2). This suggests that fluorescent BCN-modified polypeptides may be obtained when polypeptides with terminal structures GSHY or GSHYx2 are used as substrates, as in the case of polypeptides with terminal structure Y.

[0162] In the lane where the copper ion-added sample was applied, a fluorescent band was also observed on the higher molecular weight side. This band is thought to represent fusion tyrosinase bound to fluorescent BCN. From these results, it is thought that the oxidation reaction by fusion tyrosinase was promoted in the sample with added copper ions more than in the sample without added copper ions, resulting in the conversion of tyrosine residues in the fusion tyrosinase molecule to DOPA quinone residues, and that fluorescent BCN bound to these DOPA quinone residues.

Claims

1. A composition comprising a copper transport protein fusion tyrosinase, The copper transport protein fusion tyrosinase is Sequence (A): an amino acid sequence having copper transport protein function; Sequence (B): a linker sequence, and Sequence (C): Amino acid sequence with tyrosinase activity and the sequences (A) to (C) are linked in the order of (A)-(B)-(C), A composition for use in contacting the copper transport protein fusion tyrosinase with a polypeptide of interest that contains a tyrosine residue.

2. A composition for producing a modified polypeptide via said contacting, comprising: the modified polypeptide comprises a structure in which a DOPA residue and / or a DOPA quinone residue obtained by converting the tyrosine residue is bound to an organic substance having a reactive functional group, The composition according to claim 1, wherein the reactive functional group is a functional group reactive with a DOPA residue and / or a DOPA quinone residue.

3. The composition according to claim 1 or 2, wherein the sequence (A) and / or the sequence (C) is an amino acid sequence derived from a bacterium of the genus Streptomyces.

4. The composition according to claim 1 or 2, wherein the sequence (B) comprises at least one sequence selected from the group consisting of a GS linker sequence and a protease recognition sequence.

5. The copper transport protein fusion tyrosinase is Sequence (d1): the amino acid sequence represented by SEQ ID NO: 5, or Sequence (d2): an amino acid sequence having a sequence identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 5 3. The composition of claim 1 or 2, comprising:

6. 3. The composition of claim 1, wherein the target polypeptide contains a tyrosine residue within 20 amino acid residues from the N-terminus and / or within 20 amino acid residues from the C-terminus.

7. The reactive functional group is an amino group, a thiol group, an imidazole group, an alkynyl group, or a group represented by the following general formula (I): 【Chemical 1】 3. The composition according to claim 2, wherein the at least one group is selected from the group consisting of groups represented by:

8. A method for producing a modified polypeptide, comprising: contacting a copper transport protein fusion tyrosinase with a polypeptide of interest to convert at least one tyrosine residue contained in the polypeptide of interest into a DOPA residue and / or a DOPA quinone residue; The copper transport protein fused tyrosinase Sequence (A): an amino acid sequence having copper transport protein function; Sequence (B): a linker sequence, and Sequence (C): Amino acid sequence with tyrosinase activity and the sequences (A) to (C) are linked in the order of (A)-(B)-(C).

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