Single-chain polypeptide activated upon use

Abstract

The present invention provides a single-chain polypeptide that is highly activated, detoxified, and can be manufactured more safely and conveniently after reaching a specific site of use, as well as its corresponding use, the nucleic acid encoding it, and a method for producing the same. The single-chain polypeptide comprises a first domain, an intermediate amino acid sequence region, and a second domain, wherein the intermediate amino acid sequence region is located between the first and second domains, and the sequence of the intermediate amino acid sequence region includes at least one first enzymatic cleavage site, the first enzymatic cleavage site is designed to be highly specificly cleaved by a first protease present in a specific site in the human body, and the single-chain polypeptide is converted into an activated form that can suppress the release of signaling molecules by cleaving the first enzymatic cleavage site by the first protease.

Claims

1. It is a single-chain polypeptide, It comprises a first domain, an intermediate amino acid sequence region, and a second domain. Here, The aforementioned intermediate amino acid sequence region is located between the first domain and the second domain. A single-chain polypeptide characterized in that the intermediate amino acid sequence region includes at least one first-enzyme cleavage site, the first-enzyme cleavage site is designed to be cleaved with high degree of specificity by a first-enzyme protease present in a specific site in the human body, and the single-chain polypeptide is cleaved at the first-enzyme cleavage site by the first-enzyme protease to be converted into an activated form that can suppress the release of signaling molecules.

2. The single-chain polypeptide according to claim 1, characterized in that the first domain has a molecular weight of approximately 50 kDa, the second domain has a molecular weight of approximately 100 kDa, and the first domain has cleavage activity.

3. The aforementioned information material is an intercellular information material, Preferably, it is one of a neurotransmitter, an endocrine hormone, a local chemical mediator, and a gaseous signaling molecule. More preferably, the single-chain polypeptide according to claim 1 or 2, wherein the information substance is acetylcholine.

4. The aforementioned first domain is a non-toxic cytoprotease or fragment thereof capable of cleaving proteins in the exocytosis fusion apparatus of target cells; The aforementioned second domain has a binding portion and a transport portion, The binding portion is capable of binding to the target cell receptor and enters the endosome of the target cell via receptor-mediated endocytosis, and the transport portion transports the first domain to the cytosol of the target cell; Preferably, the first enzyme cleavage site is located between the first domain and the transport portion, and / or the binding portion has a carboxyl terminus and a molecular weight of about 50 kD, and the transport portion has an amino terminus and a molecular weight of about 50 kD; More preferably, the single-chain polypeptide according to any one of claims 1 to 3, wherein the protein of the exocytosis fusion apparatus is a neuronal SNARE protein, and / or the target binding site is a receptor on the presynaptic membrane of a neuron.

5. The first domain includes at least a portion of the Clostridium neurotoxin L chain, Preferably, the Clostridium neurotoxin L chain portion is a neurotoxin L chain of any one of Clostridium botulinum subtype A, B, C, D, or E or derived therefrom, and / or, the first domain comprises an amino acid sequence having at least 35%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 99%, or 100% identity with SEQ ID NO: 10, or comprises an amino acid sequence having at least 35%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 99%, or 100% identity with any one of 11 to 16, from which naturally occurring enzymatic cleavage sequences have been removed; and / or The second domain comprises at least a portion of a Clostridium neurotoxin heavy chain, preferably the portion of the Clostridium neurotoxin heavy chain being a heavy chain of any one of the following: Clostridium botulinum subtype A, Clostridium botulinum subtype B, Clostridium botulinum subtype C, Clostridium botulinum subtype D, Clostridium botulinum subtype E, Clostridium botulinum subtype F, Clostridium botulinum subtype G, Clostridium baratii, and Clostridium butyricum; or the portion of the Clostridium neurotoxin heavy chain being a heavy chain of Clostridium tetanus (C. tetanus) or a heavy chain derived therefrom, and / or The single-chain polypeptide according to any one of claims 1 to 4, characterized in that the second domain contains an amino acid sequence having at least 35%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 99%, or 100% identity with any one of sequence numbers 17 to 23.

6. The number of amino acids in the linker (L1) between the first domain and the intermediate amino acid sequence region is 0 to 20, or 0 to 10, or 0 to 5, and / or The single-chain polypeptide according to any one of claims 1 to 7, characterized in that the number of amino acids in the linker (L2) between the intermediate amino acid sequence region and the second domain is 0 to 20, or 0 to 10, or 0 to 5.

7. The aforementioned first protease is not a protease found in the digestive tract of the human body. Preferably, the specific body part is human skin tissue, and / or at least one of the first proteases is Selected from one or more of the following: epidermal-specific kallikrein (KLKs) proteases (e.g., KLK5, KLK6, KLK7), SASPase proteases, caspases (e.g., caspase-14), furin proteases, and mutants retaining the respective enzymatic activity of each; and / or, The first enzyme cleavage site includes an amino acid sequence having at least 50%, 60%, 85%, 90%, 99%, or 100% identity with any one of SEQ ID NOs: 1-4, SEQ ID NO: 50, and SEQ ID NO:

51. Hereinafter, in Sequence ID No. 50 and Sequence ID No. 51, "X" in RXXR and RRXX is any amino acid, preferably XX in RXXR is RK, TK or TR; and "XX" in RRXX is SV or KR, as described in any one of claims 1 to 6.

8. moreover, It comprises one or more of the following: a signal peptide that increases expression levels, a quantitative protein for quantification, and a tag protein. Furthermore, for one or more of the signal peptide, the quantitative protein, and the tag protein, respectively, The signal peptide comprises an amino acid sequence having at least 80%, 85%, 90%, 99%, or 100% identity with SEQ ID NO: 24: The aforementioned quantified protein is a fluorescent protein; The single-chain polypeptide according to any one of claims 1 to 7, characterized in that the tag protein is limited to one or more types of His tag, GST tag, MBP tag, and Strip-tagII tag.

9. moreover, A single-chain polypeptide according to any one of claims 1 to 8, characterized in that it includes two secondary cleavage sites located at the N-terminus of the first domain and the C-terminus of the second domain, and the secondary protease that cleaves the secondary cleavage sites cannot cleave the first cleavage site.

10. The single-chain polypeptide according to any one of claims 1 to 9, characterized in that the first enzyme cleavage site and / or the second enzyme cleavage site are not normally cleaved by proteases that come into contact during the process of expressing the single-chain polypeptide in a host cell, or during the process of synthesizing the single-chain polypeptide in an in vitro synthesis system involving cell extracts derived from host cells.

11. The second enzyme cleavage site is designed to have a high degree of specificity for the second protease. Preferably, the second protease is selected from non-human intestinal kinase, tobacco etch virus protease, protease derived from Bacillus subtilus, protease derived from amyloid-degrading bacteria (Bacillus amyliquifaciens), protease derived from rhinovirus, papain, a homolog of insect papain or a homolog of crustacean papain, and / or The single-chain polypeptide according to claim 9 or 10, characterized in that the second enzyme cleavage site includes an amino acid sequence having at least 85%, 90%, 99%, or 100% identity with any one of SEQ ID NOs: 5 to 9.

12. The sequence of the intermediate amino acid sequence region further comprises XTXS, where X is not a K amino acid, but preferably a Q amino acid; and / or The structure of the aforementioned intermediate amino acid sequence region is as follows, from the N-terminus to the C-terminus: The single-chain polypeptide according to any one of claims 1 to 11, characterized in that XTXS-linker(L3) is at least one primary enzymatic cleavage site, and the number of amino acids in L3 is 0 to 20, or 0 to 10, or 0 to 5, and furthermore, there is one primary enzymatic cleavage site.

13. The single-chain polypeptide according to any one of claims 1 to 12, characterized in that, if the second domain contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 17, the lysine at the site corresponding to position 423 of SEQ ID NO: 17 in the amino acid sequence of the second domain is mutated to non-lysine.

14. The first domain, the intermediate amino acid sequence region, and the second domain are linked from the N-terminus to the C-terminus; and / or The single-chain polypeptide according to any one of claims 1 to 13, characterized in that, if there are multiple first enzyme cleavage sites, the multiple first enzyme cleavage sites are sequentially linked from the N-terminus to the C-terminus, and all of the first enzyme cleavage sites have the same cleavage site, or each first enzyme cleavage site is different, and the number of amino acids in the linker (L4) between the multiple cleavage sites is 0 to 20, or 0 to 10, or 0 to 5.

15. Use of a single-chain polypeptide according to any one of claims 1 to 14, wherein the use includes one or more of the following: skin care, cosmetic surgery, body shaping, and treatment by muscle relaxation or relief of muscle spasms.

16. The use of a single-chain polypeptide according to any one of claims 1 to 14 in the manufacture of a product for a specific use or a pharmaceutical product, The aforementioned specific uses include one or more of the following: skincare, cosmetic surgery, body shaping, and treatment by muscle relaxation or relief of muscle spasms.

17. A single-chain polypeptide product or pharmaceutical, comprising a single-chain polypeptide as described in any one of claims 1 to 14, Preferably, the uses of the product or pharmaceutical include one or more of the following: skin care, cosmetic surgery, body shaping, and treatment by muscle relaxation or relief of muscle spasms. Alternatively, preferably, the product or pharmaceutical is a single-chain polypeptide product or pharmaceutical characterized by being used by being applied, sprayed, applied externally, or injected into a specific site of the human body.

18. A nucleic acid characterized by comprising a nucleotide sequence encoding a single-chain polypeptide as described in any one of claims 1 to 14.

19. A vector characterized by comprising the nucleic acid described in claim 18.

20. A host cell characterized by incorporating the nucleic acid described in claim 18 and / or comprising the vector described in claim 19.

21. The host cell according to claim 20, characterized in that the host cell is derived from a prokaryotic or eukaryotic cell, and further selected from the group consisting of one or more types of Escherichia coli cells, human cells, Chinese hamster ovary cells, insect cells, wheat germ cells, rabbit reticulocytes, and yeast cells.

22. The host cell according to claim 21, wherein the host cell is selected from yeast cells, and further, the yeast cell is selected from a combination of one or more species of Saccharomyces cerevisiae and Kluyveromyces yeasts, and in another preferred example, the Kluyveromyces yeast is selected from a combination of one or more species of Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces dobzhanskii.

23. An in vitro cell-free synthesis system, An in vitro cell-free synthesis system characterized by synthesizing a single-chain polypeptide using mRNA or DNA encoding a single-chain polypeptide as described in any one of claims 1 to 14 as a template.

24. The in vitro cell-free synthesis system according to claim 23, characterized by comprising one or more of the following: (1) mRNA or DNA template encoding the single-chain polypeptide; (2) A cell extract, wherein the cell extract is a yeast cell extract, and further, the cell extract is derived from one or more of the following: Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces yeast, preferably the yeast cell extract is derived from Kluyveromyces yeast, and more preferably from Kluyveromyces lactis yeast; (3) Any one or more of the following ingredients: Amino acid mixture, dNTPs, RNA polymerase, DNA polymerase, energy supply system, polyethylene glycol, and aqueous solvent.

25. A method for producing a single-chain polypeptide according to any one of claims 1 to 14, Using mRNA or DNA encoding a single-chain polypeptide according to any one of claims 1 to 14 as a template, an in vitro synthesis reaction is carried out to obtain the single-chain polypeptide, Preferably, the in vitro synthesis reaction is carried out in a reaction system including the in vitro cell-free synthesis system described in claim 23 or 24. Furthermore, the method for producing a single-chain polypeptide is characterized in that the volume ratio of the DNA template to other components involved in the in vitro synthesis reaction is in the range of 1:10 to 1:50, 1:20 to 1:40, 1:25 to 1:35, or 1:

30.

26. The cell-free in vitro synthesis system comprises yeast cell extract, amino acid mixture, dNTPs, RNA polymerase, DNA polymerase, energy supply system, polyethylene glycol, and aqueous solvent; moreover, The method for producing a yeast cell extract according to claim 25, characterized in that the yeast cell extract is derived from one or more of the following: Saccharomyces cerevisiae, Pichia yeast, and Clyveromyces yeast, preferably derived from Clyveromyces yeast, and more preferably from Clyveromyces lactis yeast.

27. The manufacturing method according to claim 26, characterized in that the yeast cell extract accounts for 50 to 80% of the total volume of the cell-free extracellular protein synthesis system.

28. Furthermore, the manufacturing method according to any one of claims 25 to 27 is characterized by comprising cleaving the obtained single-chain polypeptide containing the second enzyme cleavage site with a second protease to obtain a single-chain polypeptide containing only the first domain, the intermediate amino acid sequence region, and the second domain.

29. The production of a single-chain polypeptide according to any one of claims 1 to 13, using the nucleic acid according to claim 18, the vector according to claim 19, the host cell according to any one of claims 20 to 22, and the in vitro cell-free synthesis system according to claim 23 or 24.

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