Secretory fusion protein, fusion protein, nucleic acid, expression vector, transformant, and method for producing fusion protein

Abstract

This secretory fusion protein contains: a secretion signal sequence; a modified peptide that attenuates the cell permeation ability of a heparan sulfate-binding peptide and maintains the heparan sulfate-binding ability; and a target protein.

Description

Secretory fusion protein, fusion protein, nucleic acid, expression vector, transformant, and method for producing fusion protein

[0001] The present disclosure relates to a secretory fusion protein, a fusion protein, a nucleic acid, an expression vector, a transformant, and a method for producing a fusion protein.

[0002] Heparan sulfate is a linear polysaccharide having a backbone in which disaccharide units composed of glucuronic acid and N-acetylglucosamine are repeated, and each sugar residue has various structures by being sulfated in various patterns. In addition, heparan sulfate proteoglycan is a group of molecules in which heparan sulfate is covalently bonded to a core protein, and is present on the cell membrane surface and in the extracellular matrix. As heparan sulfate proteoglycan, syndecan, glypican, agrin, etc. are known. Heparan sulfate constituting these heparan sulfate proteoglycans is known to promote cell proliferation activity by growth factors such as fibroblast growth factor by forming a complex with the receptor of the growth factor.

[0003] That is, growth factors and receptors that form a complex with heparan sulfate include heparan sulfate-binding membrane-permeable peptides that interact with heparan sulfate. Heparan sulfate-binding membrane-permeable peptides have been identified in various proteins as described in Non-Patent Document 1. Non-Patent Document 1 discloses the amino acid sequence of the identified heparan sulfate-binding membrane-permeable peptide and the amino acid sequence of the heparan sulfate-binding domain in the heparan sulfate-binding membrane-permeable peptide.

[0004] In addition, Patent Document 1 discloses a peptide consisting of a specific amino acid sequence as a heparan sulfate-binding peptide having a hydrophobic sequence. The heparan sulfate-binding peptide disclosed in Patent Document 1 is said to have a higher heparin-neutralizing activity and a potentially higher affinity for glycosaminoglycan and proteoglycan compared to those without a hydrophobic sequence. Patent Document 1 discloses the use of the heparan sulfate-binding peptide as a medicine.

[0005] Incidentally, heparan sulfate-binding membrane-permeable peptides are a type of membrane-permeable peptide. Therefore, recombinant proteins containing such heparan sulfate-binding membrane-permeable peptides are produced intracellularly and are difficult to secrete extracellularly. For example, Non-Patent Document 1 discloses a technique for improving the intracellular productivity of recombinant proteins to which membrane-permeable peptides have been added.

[0006] Patent Document 1: U.S. Patent No. 7,259,140

[0007] Non-patent document 1: Chien-Jung Chen et al., BioMed Research International, Volume 2015, Article ID 237969. Non-patent document 2: Zifan Gong et al., Mol. Biotechnol. (2016), 58:838-849.

[0008] Generally, when producing recombinant proteins, secreting them extracellularly from transformed cells is simpler and more efficient than producing them intracellularly. This is because intracellular production of recombinant proteins requires steps such as cell lysis, isolation of the recombinant protein from impurities including the cell lysis product, and purification. However, it has been difficult to secrete recombinant proteins with membrane-permeable peptides, such as heparan sulfate-binding membrane-permeable peptides, extracellularly.

[0009] As described above, there has been a problem in that recombinant proteins having heparan sulfate binding ability cannot be produced simply and efficiently. Therefore, the object of this disclosure is to provide a secreted fusion protein having heparan sulfate binding ability, a fusion protein having heparan sulfate binding ability, a nucleic acid encoding the secreted fusion protein, an expression vector having the nucleic acid, a transformant, and a method for producing the fusion protein.

[0010] As a result of diligent research to achieve the above objective, the present inventors have found that by using a modified peptide in which the cell permeability of the heparan sulfate-binding peptide is weakened while the heparan sulfate-binding ability is maintained, a fusion protein having heparan sulfate-binding ability can be secreted and produced extracellularly, thus completing this disclosure. Specifically, one aspect of this disclosure is as follows: <1> A secretory fusion protein comprising a secretory signal sequence, a modified peptide in which the cell permeability of the heparan sulfate-binding peptide is weakened while the heparan sulfate-binding ability is maintained, and a target protein. <2> The secretory fusion protein according to <1>, wherein the modified peptide is the heparan sulfate-binding domain of the heparan sulfate-binding peptide. <3> The secretory fusion protein according to <1> or <2>, wherein the modified peptide has 75% or less basic amino acid residues among all amino acid residues constituting the modified peptide. <4> The modified peptide is a secretory fusion protein according to <1> or <2>, wherein 50% or less of the total amino acid residues constituting the modified peptide are basic amino acid residues. <5> The modified peptide is a secretory fusion protein according to any one of <1> to <4>, wherein the modified peptide is one amino acid sequence selected from the group consisting of (a) to (d) below: (a) KTRYKARRA (SEQ ID NO: 1) (b) TRRQRT (SEQ ID NO: 2) (c) SRPRRP (SEQ ID NO: 3) (d) NRRMKWKK (SEQ ID NO: 30) <6> The target protein is a secretory fusion protein according to any one of <1> to <5>, wherein the target protein interacts with a protein present on the cell surface. <7> The secretory fusion protein according to <6>, wherein the protein present on the cell surface is a receptor, and the target protein is an agonist to the receptor. <8> The secretory fusion protein according to <7>, wherein the receptor is a receptor with which a growth factor interacts.<9> The growth factors include EGF, GM-CSF, M-CSF, IL-6, PDGF-AA, PDGF-BB, IL-2, FGF-2, IL-3, IL-7, TGF-β1, FLt3-Ligand, IL-4, TPO, I L-5, IL-15, IL-21, IL-18, IFN-γ, SCF, VEGF165, BMP-4, TNF-alpha, Noggin, R-Spondin, Wnt-3a, FGF-10, KGF, Activin A secreted fusion protein as described in <8>, which is a growth factor selected from the group consisting of A, IGF-1, NRG1, Jaggerd1, VEGF121, HGF, BMP-2, Shh, FGF-8, BDNF, GDNF, Artemin, Beta-NGF, CDNF, CNTF, GMF-beta, MANF, Midkine, NT-3, NT-4, NGF, Pleiotropin, TrkA, TrkB, and TrkC. <10> The target protein is a secreted fusion protein as described in any one of <1> to <9>, which has a pair of interaction sites with a protein present on the cell surface. <11> The secretory fusion protein according to <10>, wherein the interaction site is a small molecule antibody selected from the group consisting of a single-chain antibody (scFv) and a heavy chain antibody variable region (VHH) antibody that specifically binds to the protein. <12> The secretory fusion protein according to any one of <1> to <11>, wherein the linker is a flexible linker. <13> The secretory fusion protein according to <12>, wherein the linker is a flexible linker. <14> The secretory fusion protein according to <13>, wherein the flexible linker contains an amino acid sequence of 5 amino acid residues or less, including 2 or more glycine residues. <15> The secretory fusion protein according to any one of <1> to <14>, wherein the secretory signal sequence is a cleavage-promoting sequence between the secretory signal sequence and the modified peptide or the target protein. <16> The secretory fusion protein according to <15>, wherein the cleavage-promoting sequence is A or AQ. <17> A secretory fusion protein according to any one of <1> to <16>, further having a purification tag sequence at its C-terminus. <18> A secretory fusion protein according to <17>, wherein the modified peptide is linked to the tag sequence directly or via a linker.<19> The linker is an amino acid sequence consisting of amino acids other than glycine, the secreted fusion protein described in <18>. <20> A fusion protein obtained by cleaving a region containing a secretion signal sequence from the secreted fusion protein described in any one of <1> to <19>. <21> A nucleic acid encoding the secreted fusion protein described in any one of <1> to <19>. <22> An expression vector having the nucleic acid described in <21>. <23> A transformant having the nucleic acid described in <21>. <24> A method for producing a fusion protein, comprising the steps of culturing the transformant described in <23> and recovering the fusion protein described in <20> from the culture medium in which the transformant was cultured.

[0011] According to one aspect of this disclosure, it is possible to provide a secretory fusion protein capable of secreting and producing a fusion protein having heparan sulfate binding ability outside of cells, the secreted fusion protein, a nucleic acid encoding the secretory fusion protein, an expression vector having the nucleic acid, a transformant, and a method for producing the fusion protein.

[0012] Figure 1 is a schematic diagram showing the in vivo behavior of the fusion proteins of this disclosure. Figure 2 is an electrophoresis image showing the results of Western blotting for the secreted fusion proteins of Examples 1 to 4 and Comparative Example 1. Figure 3 is an electrophoresis image showing the results of Western blotting for the secreted fusion proteins of Examples 1 and 5 to 10. Figure 4 is an electrophoresis image showing the results of Western blotting for the secreted fusion proteins of Examples 5, 6 and Comparative Example 1 after purification. Figure 5 is a characteristic diagram showing the results of evaluating the heparan sulfate binding ability of the secreted fusion proteins of Examples 5, 6 and Comparative Example 1. Figure 6 is an electrophoresis image showing the results of Western blotting for the secreted fusion proteins of Examples 5, 6 and 11 to 14. Figure 7 is a characteristic diagram showing the results of evaluating the heparan sulfate binding ability of the secreted fusion proteins of Examples 5 and 13. Figure 8 is a characteristic diagram showing the results of evaluating the heparan sulfate binding ability of the secreted fusion proteins of Examples 6 and 14. Figure 9 is an electrophoresis image showing the results of Western blotting for the secreted fusion proteins of Example 14 and Comparative Example 2.

[0013] In explaining this disclosure, specific examples will be given, but the content is not limited to the following, as long as it does not deviate from the intent of this disclosure, and may be modified as appropriate.

[0014] In this disclosure, a numerical range expressed using "~" means a range that includes the numbers written before and after "~" as the lower and upper limits. In numerical ranges described in stages in this disclosure, the upper or lower limit stated in one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Also, in numerical ranges described in this disclosure, the upper or lower limit of that numerical range may be replaced with the values ​​shown in the examples. In this disclosure, the amount of each component in a composition means the total amount of multiple substances present in the composition if there are multiple substances corresponding to each component in the composition, unless otherwise specified. In this disclosure, a preferred combination of embodiments means a more preferred embodiment. In this disclosure, the term "process" includes not only independent processes but also processes that are not clearly distinguishable from other processes, as long as their intended purpose is achieved.

[0015] In this disclosure, "protein-coding nucleic acid" means a nucleotide chain or its complementary chain containing a base sequence designed based on a codon table for the amino acid sequence of a protein, and such nucleotide chains or their complementary chains, such as polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA, etc., are included in the meaning of "protein-coding nucleic acid." Such nucleic acids are single-stranded, double-stranded, or triple-stranded or more nucleotides, and associations of DNA and RNA chains, nucleotides on a single nucleotide chain containing both ribonucleotides (RNA) and deoxyribonucleotides (DNA), and double-stranded or triple-stranded or more nucleotides containing such nucleotide chains are also included in the meaning of "nucleic acid." Note that base sequence and nucleotide sequence are synonymous.

[0016] In this disclosure, "peptide" refers to a structure in which multiple amino acids are linked by peptide bonds (-CO-NH-). Depending on the number of linked amino acids, they are called dipeptides, tripeptides, oligopeptides, polypeptides, etc., but in this disclosure, they are collectively referred to as peptides regardless of the number of amino acids. Therefore, in this disclosure, when the term "peptide" is used, it also includes proteins.

[0017] <Secreted Fusion Protein> The secreted fusion protein of this disclosure contains a secretion signal sequence, a modified peptide in which the cell permeability of the heparan sulfate-binding peptide is attenuated while the heparan sulfate-binding ability is maintained, and a target protein. Because the secreted fusion protein of this disclosure contains a secretion signal sequence, it can permeate the cell membrane and is secreted as a fusion protein containing the modified peptide and the target protein. When the secreted fusion protein of this disclosure is biosynthesized within a cell and secreted across the cell membrane, the secretion signal is cleaved, and it is secreted as a fusion protein containing the modified peptide and the target protein. Because the secreted fusion protein contains a modified peptide in which the cell permeability of the heparan sulfate-binding peptide is attenuated, it is presumed to exist extracellularly without being taken up again into the cell.

[0018] The secretory fusion protein of this disclosure may have a sequence in which a secretory signal sequence, a modified peptide, and a target protein are arranged in this order from the N-terminus to the C-terminus, or a sequence in which a secretory signal sequence, a target protein, and a modified peptide are arranged in this order. In other words, the secretory fusion protein of this disclosure may contain multiple modified peptides or multiple target proteins.

[0019] (Modified Peptides) In this disclosure, a modified peptide means a heparan sulfate-binding peptide in which the cell permeability is weakened while the heparan sulfate binding ability is maintained. Weakening of cell permeability means that the cell permeability inherent in the heparan sulfate-binding peptide is reduced or lost. Heparan sulfate-binding peptides are a type of cell permeable peptide and possess cell permeability due to a region containing basic amino acids selected from the group consisting of lysine, arginine, and histidine. Therefore, the cell permeability of a heparan sulfate-binding peptide can be weakened by deleting some of the basic amino acids contained in the above region of the heparan sulfate-binding peptide, or by modifying some of the basic amino acids to amino acids other than basic amino acids. However, if all of the basic amino acids contained in the above region of the heparan sulfate-binding peptide are deleted, or if all of the basic amino acids are modified to amino acids other than basic amino acids, there is a risk that not only cell permeability but also heparan sulfate binding ability will be lost. In this disclosure, the heparan sulfate binding ability can be maintained by deleting some of the basic amino acids in the above region of the heparan sulfate binding peptide, or by modifying the peptide by replacing them with amino acids other than basic amino acids. Examples of amino acids other than basic amino acids include glutamine, glutamic acid, threonine, tyrosine, alanine, asparagine, aspartic acid, valine, serine, methionine, proline, cysteine, leucine, isoleucine, glycine, tryptophan, and phenylalanine.

[0020] In the secreted fusion protein of this disclosure, the modified peptide is preferably positioned N-terminally relative to the target protein. When the modified peptide is positioned N-terminally relative to the target protein, the heparan sulfate binding ability of the modified peptide is increased. However, even if the modified peptide is positioned C-terminally relative to the target protein in the secreted fusion protein of this disclosure, the heparan sulfate binding ability of the modified peptide can be sufficiently maintained.

[0021] The cell permeability of heparan sulfate-conjugated peptides can be measured, for example, according to the method described in BioMed Research International, Volume 2015, Article ID 237969. Specifically, the cell permeability of heparan sulfate-conjugated peptides can be measured by using a cell line expressing heparan sulfate on the cell surface, a heparan sulfate-conjugated peptide labeled with a fluorescent dye, an anti-heparan sulfate antibody, and a fluorescently labeled antibody for detecting the anti-heparan sulfate antibody, and measuring the fluorescence derived from the fluorescently labeled antibody for detecting the anti-heparan sulfate antibody using flow cytometry or a fluorescence microscope. In this disclosure, "reduced cell permeability" means that, when the cell permeability of the original heparan sulfate-bound peptide is set to 100% as described above, the cell permeability of the modified peptide is 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less, or 0%.

[0022] Furthermore, the heparan sulfate binding ability of a heparan sulfate-binding peptide can be measured as follows: Biotinylated heparan sulfate is immobilized on a streptavidin-coated ELISA plate, a protein containing a heparan sulfate-binding peptide is reacted with it, and the reaction is detected with an HRP (Horseradish peroxidase) labeled antibody that reacts with the protein. This method allows for the measurement of the heparan sulfate-binding ability of the tested heparan sulfate-binding peptide. In this disclosure, maintaining heparan sulfate-binding ability means that, when the heparan sulfate-binding ability of the original heparan sulfate-binding peptide measured as described above is taken as 100%, the heparan sulfate-binding ability of the modified peptide is 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more, or 100%.

[0023] In this disclosure, the modified peptide can be, for example, the heparan sulfate binding domain in a heparan sulfate binding peptide. That is, the heparan sulfate binding domain contained in a known heparan sulfate binding peptide or a novel heparan sulfate binding peptide can be modified and used as the modified peptide in this disclosure. In heparan sulfate binding peptides, the region rich in basic amino acids is usually involved in both cell permeability and heparan binding. Therefore, as the modified peptide in this disclosure, the heparan sulfate binding domain rich in these basic amino acids can be modified and used in a heparan sulfate binding peptide.

[0024] Furthermore, in this disclosure, the modified peptide preferably has 75% or less basic amino acid residues among all amino acid residues constituting the modified peptide, more preferably 55% or less, and even more preferably 50% or less. By setting the basic amino acid residues among all constituent amino acid residues of the modified peptide to this range, it is possible to further reduce cell permeability while maintaining the ability to bind to heparan sulfate. In particular, the modified peptide can be a peptide consisting of 5 to 20 amino acids in total length, preferably a peptide consisting of 5 to 17 amino acids in total length, more preferably a peptide consisting of 5 to 15 amino acids in total length, and even more preferably a peptide consisting of 5 to 10 amino acids in total length. By setting the total length of the modified peptide to this range and setting the basic amino acid residues among all amino acid residues to the above range, it is possible to further reduce cell permeability while maintaining the ability to bind to heparan sulfate.

[0025] In this disclosure, the modified peptides can be designed based on known heparan sulfate-conjugated peptides. Examples of known heparan sulfate-conjugated peptides include those described in BioMed Research International, Volume 2015, Article ID 237969. Table 1 shows examples of known heparan sulfate-conjugated peptides.

[0026]

[0027] In this disclosure, the modified peptide may be a peptide consisting of the amino acid sequence described in the "Heparan Sulfate Binding Domain" column in Table 1. In particular, in this disclosure, the modified peptide is preferably a peptide consisting of one amino acid sequence selected from the group consisting of (a) to (d) below: (a) KTRYKARRA (SEQ ID NO: 1) (b) TRRQRT (SEQ ID NO: 2) (c) SRPRRP (SEQ ID NO: 3) (d) NRRMKWKK (SEQ ID NO: 30) Modified peptides defined by these specific amino acid sequences have reduced cell permeability compared to the original heparan sulfate binding peptide, while maintaining their ability to bind to heparan sulfate.

[0028] (Secretion Signal Sequence) The secretory fusion protein of this disclosure is equipped with a secretory signal sequence (sometimes simply referred to as a signal sequence or signal peptide). In the secretory fusion protein of this disclosure, the secretory signal sequence is preferably located at the N-terminus. The secretory fusion protein of this disclosure is secreted extracellularly due to the presence of the secretory signal sequence. When the secretory fusion protein of this disclosure is biosynthesized intracellularly, the secretory signal sequence is recognized by SRP (Signal Recognition Particle) and guided to the endoplasmic reticulum membrane via the SRP receptor (SR). Subsequently, the secretory signal sequence is cleaved from the secretory fusion protein of this disclosure and secreted extracellularly through membrane permeability channels. In other words, the secretory fusion protein of this disclosure has a secretory signal sequence when biosynthesized intracellularly, but does not have a secretory signal sequence when secreted extracellularly. In this disclosure, the secretory fusion protein biosynthesized intracellularly is secreted extracellularly and exists as a protein that does not have a secretory signal sequence. In this disclosure, proteins that do not have this secretion signal sequence are referred to as "fusion proteins."

[0029] A secretory signal sequence is an extracellular transit signal necessary for the secretion of proteins biosynthesized by gene expression into the extracellular space. A secretory signal sequence has a primary structure in which positively charged amino acids such as lysine and arginine are positioned at the N-terminus, followed by a sequence of highly hydrophobic amino acids such as alanine, leucine, valine, isoleucine, and phenylalanine. Furthermore, it is preferable to select an appropriate secretory signal sequence depending on the host cell that biosynthesizes the secretory fusion protein. For example, if the host cell is E. coli, a secretory signal sequence derived from E. coli is preferable; if the host cell is Bacillus subtilis, a secretory signal sequence derived from Bacillus subtilis is preferable; if the host cell is Corynebacterium, a secretory signal sequence derived from Corynebacterium is preferable; and if the host cell is yeast, a secretory signal sequence derived from yeast is preferable.

[0030] In this disclosure, the secretory signal sequence is not particularly limited, and any conventionally known secretory signal sequence can be used. The secretory signal sequence may be derived from a gene expressed in a prokaryote or a gene expressed in a eukaryote. For example, examples of secretory signal sequences include those derived from the Pelb gene, OmpA gene, DsbA gene, TorA gene, MalE gene, StII gene, PhoA gene, PhoE gene, LamB gene, AmyE gene, AprE gene, NprE gene, LipA gene, CspB gene, CspA gene, PorB gene, Ncgl337 gene, MFα1 gene, SUC2 gene, PHO5 gene, etc.

[0031] Furthermore, the secreted fusion protein of this disclosure preferably has a cleavage-promoting sequence between the secretory signal sequence located at the N-terminus and the region extending from the secretory signal sequence toward the C-terminus (modified peptide or target protein). The cleavage-promoting sequence refers to an amino acid sequence recognized by a signal peptidase that has the activity to cleave the secretory signal sequence. Examples of cleavage-promoting sequences include A or AQ (A: alanine, Q: glutamine). By having a cleavage-promoting sequence between the secretory signal sequence and the region extending from the secretory signal sequence toward the C-terminus (modified peptide or target protein), the secreted fusion protein of this disclosure can be secreted more efficiently outside the cell, and degradation products cleaved outside the secretory signal sequence and the region extending from the secretory signal sequence toward the C-terminus are reduced, enabling high production of the target fusion protein outside the cell.

[0032] (Target Protein) In this disclosure, the target protein is not particularly limited and may be any protein. According to this disclosure, the target protein is secreted extracellularly as a fusion protein formed by fusing a modified peptide in which the cell permeability of the heparan sulfate-binding peptide is weakened while the heparan sulfate-binding ability is maintained. That is, since the fusion protein contains both the modified peptide and the target protein, it can come into close proximity with molecules containing heparan sulfate in vivo due to the function of the modified peptide. As a result, the fusion protein can exhibit various physiological activities in vivo depending on the function of the target protein. Therefore, in this disclosure, the target protein is preferably a protein whose function is activated by coming into close proximity with heparan sulfate or molecules containing heparan sulfate.

[0033] Here, molecules containing heparan sulfate include heparan sulfate itself and heparan sulfate proteoglycans (HSPGs). Heparan sulfate proteoglycans are a group of molecules in which heparan sulfate is covalently bonded to a core protein, and they are present on the cell membrane surface and in the extracellular matrix of animal cells. Examples of heparan sulfate proteoglycans include syndecan, glypican, agrin, and perlecan. Syndecan includes four types, syndecan 1 to syndecan 4, which are present on the cell surface via transmembrane domains. Glypican includes six types, glypican 1 to glypican 6, which are present on the cell surface via GPI (glycosyl-phosphatidyl-inositol). Perlecan and agrin are proteoglycans that are secreted extracellularly and become part of the extracellular matrix.

[0034] In this disclosure, the target protein can be a protein that interacts with a protein present on the cell surface. In this case, the fusion protein of this disclosure can bring a protein present on the cell surface and a molecule containing heparan sulfate (e.g., a heparan sulfate proteoglycan such as syndecane) into close proximity in vivo. Thus, the fusion protein can bring the cell surface protein that the target protein interacts with and the molecule containing heparan sulfate to which the modified peptide binds into close proximity in vivo, and can exhibit various physiological activities depending on the function of the cell surface protein.

[0035] In this disclosure, a protein present on the cell surface with which the target protein interacts is referred to as a "receptor," and the target protein can be an agonist for said receptor. A receptor is a substance that receives signals from outside the cell, and usually refers to a substance that transmits signals through the interaction of ligands. Such receptors are not particularly limited and include those that use cytokines as ligands, growth factors (also called proliferation factors) as ligands, and hormones as ligands. The target protein preferably functions as an agonist that acts on these receptors to enhance signal transduction from the receptor. In this disclosure, the target protein may act as a so-called partial agonist, or it may act as an antagonist.

[0036] In particular, in this disclosure, the receptor is preferably a receptor with which growth factors interact. That is, the fusion protein of this disclosure is preferably such that the target protein acts on a receptor with which growth factors interact and functions as an agonist. The growth factors are not particularly limited, but include EGF, GM-CSF, M-CSF, IL-6, PDGF-AA, PDGF-BB, IL-2, FGF-2, IL-3, IL-7, TGF-β1, FLt3-Ligand, IL-4, TPO, IL-5, IL-15, IL-21, IL-18, IFN-γ, SCF, VEGF165, BMP-4, TNF-alpha, Noggin, R-Spondin, Wnt-3a, FGF-10, KGF, Activin A, IGF-1, NRG1, and Jagged Examples of growth factors include VEGF121, HGF, BMP-2, Shh, FGF-8, BDNF, GDNF, Artemin, Beta-NGF, CDNF, CNTF, GMF-beta, MANF, Midkine, NT-3, NT-4, NGF, Pleiotropin, TrkA, TrkB, and TrkC. In the fusion protein of this disclosure, the target protein preferably acts on receptors on which these growth factors act. All of these receptors for growth factors are well known; for example, the growth factor EGF is known as the EGF receptor. The same applies to growth factors other than EGF.

[0037] In particular, examples of growth factors include FGF-2, HGF, TGF-β1, BDNF, VEGF165, Wnt-3a, TPO, PDGF-AA, KGF, IL-15, Activin A, FLt3 Ligand, IGF-1, IL-21, R-Spondin, BMP-4, GDNF, IL-2, M-CSF, SCF, GM-CSF, IL-7, and PDGF-BB. Furthermore, it is preferable that the growth factors be FGF-2, HGF, BDNF, VEGF, Wnt-3a, and TPO. In the fusion protein of this disclosure, it is even more preferable that the target protein acts on receptors on which these growth factors act.

[0038] Generally, when a growth factor interacts with its receptor, the receptor forms a dimer. The intracellular domain of the dimerized receptor is then activated, and a signal is transmitted downstream. Therefore, it is preferable that the secreted fusion protein or fusion protein of this disclosure promotes the dimerization of proteins present on the cell surface, such as receptors. Specifically, in the secreted fusion protein or fusion protein of this disclosure, the target protein preferably has a pair of interaction sites with proteins present on the cell surface. Each interaction site only needs to interact with a protein present on the cell surface, such as a receptor; for example, they can be homodimers having the same interaction sites. In the secreted fusion protein or fusion protein of this disclosure, the target protein can promote the dimerization of proteins present on the cell surface by having a pair of interaction sites with proteins present on the cell surface. Therefore, if the protein present on the cell surface is a growth factor receptor, the secreted fusion protein or fusion protein of this disclosure can promote the dimerization of that receptor.

[0039] The interaction site on the target protein is not particularly limited as long as it specifically binds to proteins present on the cell surface, and includes, for example, antibodies, nucleic acid aptamers, glycans, proteins, etc. An example of an interaction site is an antibody.

[0040] In the present disclosure, "antibody" means a protein that exhibits immunoreactivity against an antigen. The biological species from which the antibody is derived is not particularly limited. Preferably, the antibody is derived from birds and mammals. For example, chicken, ostrich, mouse, rat, guinea pig, rabbit, goat, donkey, sheep, camel, horse, or human, etc. can be mentioned. Further, an antibody includes a framework region (FR) and a complementarity-determining region (CDR). An antibody includes a single species of immunoglobulin capable of recognizing an antigen, or a recombinant antibody or a synthetic antibody including at least one set of light-chain variable region (VL region) and heavy-chain variable region (VH region) contained in an immunoglobulin. When an antibody is composed of an immunoglobulin molecule, the immunoglobulin can be of any class and any subclass.

[0041] Also, the antibody may be a chimeric antibody or a humanized antibody. Further, the antibody may also be a synthetic antibody synthesized chemically or by recombinant DNA methods. Specifically, for example, scFv (single chain Fragment of variable region: single-chain antibody) can be mentioned. In an immunoglobulin molecule, a set of variable regions (light-chain variable region VL and heavy-chain variable region VH) that form a functional antigen-binding site are located on separate polypeptide chains of a light chain and a heavy chain. scFv is a synthetic antibody having a molecular weight of about 3 kDa or less and having a structure in which VL and VH are linked by a flexible linker of sufficient length and included in one polypeptide chain in an immunoglobulin molecule. In scFv, a set of variable regions can self-assemble with each other to form one functional antigen-binding site. scFv can be obtained by incorporating the recombinant DNA encoding it into a vector and expressing it using known techniques.

[0042] Antibodies can also be modified. "Modification" here includes functional modifications necessary for antigen-specific binding activity, such as glycosylation, and labeling modifications necessary for antibody detection. Glycosylation modifications on antibodies are performed to adjust the antibody's affinity for the target antigen. Specifically, examples include modifications in the antibody's FR (free radical) where substitutions are introduced into amino acid residues constituting the glycosylation site, thereby removing the glycosylation site and causing a loss of glycosylation at that site.

[0043] In particular, in this disclosure, a small molecule antibody refers to an antibody fragment that consists of a part of a full-length antibody and exhibits an immune response to an antigen in the same way as a full-length antibody. Examples of small molecule antibodies include Fab, Fab', and F(ab'). ２ Fv fragment, Fv fragment stabilized by disulfide bonds (dsFv), (dsFv) ２ Examples include bispecific dsFv (dsFv-dsFv'), disulfide bond-stabilized diabodies (ds-diabodies), single-chain antibody molecules (scFv), dimeric scFv (bivalent diabodies), multispecific antibodies, heavy chain antibodies such as camelized single-domain antibodies (camelized antibodies; VHH (variable domain of heavy chain of heavy chain antibody) antibodies), nanobodies, domain antibodies, and bivalent domain antibodies.

[0044] In the secreted fusion protein or the target protein contained in the fusion protein of this disclosure, the interaction site is preferably a small molecule antibody that specifically binds to the receptor described above. In particular, the interaction site is more preferably one small molecule antibody selected from the group consisting of single-chain antibodies (scFv) and heavy chain antibody variable region (VHH) antibodies that specifically bind to proteins present on the cell surface. This is because these scFv and VHH antibodies have low molecular weights among small molecule antibodies and can be easily prepared by recombinant DNA method. In the secreted fusion protein or the target protein contained in the fusion protein of this disclosure, by using scFv or VHH antibodies as a pair of interaction sites, the dimerization of the receptor described above can be promoted.

[0045] In particular, in the target protein contained in the secreted fusion protein or fusion protein of the present disclosure, it is preferable that the pair of interaction sites be single-domain antibodies. A single-domain antibody means an antibody consisting of only the variable region of an antibody composed of only a heavy chain. Examples of single-domain antibodies include VHH antibodies, VNAR antibodies, and the like. In particular, in the target protein contained in the secreted fusion protein or fusion protein of the present disclosure, it is most preferable that the pair of interaction sites be VHH antibodies. A VHH antibody has four framework regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3). In a VHH antibody, these FRs and CDRs are alternately linked. The molecular weight of a VHH antibody is about 12 kDa to 15 kDa. Here, CDR refers to the so-called complementarity-determining region. In single-domain antibodies such as VHH antibodies, the antigen directly interacts with the CDR. The positions of CDR1 and CDR3 refer to the positions determined using Kabat numbering.

[0046] (Linker) The secreted fusion protein or fusion protein of the present disclosure may have the modified peptide described above and the target protein described above directly linked, but it is preferable to have a linker between the modified peptide and the target protein. That is, in the secreted fusion protein or fusion protein of the present disclosure, it is preferable that the modified peptide and the target protein are linked via a linker. By linking the modified peptide and the target protein via a linker, the modified peptide and the target protein can each exert their functions without interfering with each other.

[0047] The linker placed between the modified peptide and the target protein is not particularly limited, but is preferably a flexible linker. A flexible linker is a linker in which the linking portions can be freely positioned, and preferably contains an amino acid sequence of 5 amino acid residues or less, including two or more glycine residues. More specifically, the flexible linker may be "GGGGGS", or it may be an amino acid sequence in which "GGGGGS" is repeated 2 to 4 times as one unit.

[0048] In the secreted fusion protein or fusion protein of this disclosure, by arranging a linker, particularly a flexible linker, between the modified peptide and the target protein, at least one effect selected from the group consisting of an effect of suppressing inhibition of protein folding formation, an effect of improving protein expression levels, and an effect of reducing protein degradation products can be achieved.

[0049] (Purification Tag) The secreted fusion protein or fusion protein of this disclosure may have a purification tag sequence at its C-terminus. After the secreted fusion protein of this disclosure is biosynthesized intracellularly, the fusion protein is secreted extracellularly. Having a purification tag sequence at its C-terminus allows for easy recovery of the fusion protein of this disclosure secreted extracellularly. Here, conventionally known tag sequences can be used, such as His tag, Flag tag, Spot tag, C tag, Strip tag, GST tag, MBP tag, etc.

[0050] In the secreted fusion protein or fusion protein of this disclosure, the modified peptide described above is preferably linked to a tag sequence directly or via a linker. In this case, unlike the flexible linker described above, it is preferable that the linker is one in which the tag sequence located at the C-terminus does not move freely in space. Such a linker is not particularly limited, but can be an amino acid sequence consisting of amino acids other than glycine. As an example, it is preferable to link the modified peptide and the tag sequence via a single serine residue. In the secreted fusion protein or fusion protein of this disclosure, by linking the modified peptide to the tag sequence directly or via a linker, the negative charge of the C-terminal tag sequence is prevented from canceling the positive charge in the modified peptide, and the modified peptide can exert its function.

[0051] <In vivo behavior model> The fusion protein of this disclosure, constructed as described above, can bring proteins such as receptors present on the cell surface into close proximity with molecules containing heparan sulfate in vivo, and can exhibit various physiological activities depending on the function of the target protein in vivo. Figure 1 shows the expected in vivo behavior of the fusion protein of this disclosure. As shown in Figure 1, fusion protein 1 contains a pair of interaction sites 2A and 2B that specifically bind to receptors present on the cell surface, and a modified peptide 3 that has the ability to bind to heparan sulfate. Note that the fusion protein 1 shown in Figure 1 is simplified and omits the linker, purification tag, etc. mentioned above.

[0052] Fusion protein 1 promotes the dimerization of receptor 4 in vivo through a pair of interaction sites 2A and 2B, and also brings heparan sulfate-containing molecule 5 closer to the receptor through modified peptide 3. Examples of receptor 4 to be dimerized include the FGF receptor as described above, and examples of heparan sulfate-containing molecule 5 include syndecane as described above. Since heparan sulfate-containing molecule 5, such as syndecane, has multiple heparan sulfate molecules, other heparan sulfate molecules present on the cell surface can also be brought closer to it through heparan sulfate molecules other than the one interacting with fusion protein 1.

[0053] As described above, fusion protein 1 not only promotes the dimerization of receptor 4, but also brings molecules 5 containing heparan sulfate and other proteins 6 closer to receptor 4. This allows for the activation of the intracellular domain 7 of receptor 4. Thus, fusion protein 1 can exhibit physiological activity in vivo, such as activating the intracellular domain 7 of receptor 4.

[0054] <Nucleic Acids> The nucleic acids of this disclosure include a region encoding the secreted fusion protein of this disclosure described above. By introducing the nucleic acids of this disclosure into a suitable host and expressing the region encoding the secreted fusion protein, the secreted fusion protein can be biosynthesized within the host, and the fusion protein of this disclosure can be secreted into a culture medium. The base sequence of such nucleic acids can be appropriately designed based on the amino acid sequence and codon table of the secreted fusion protein described above. The optimal codon can be selected according to the frequency of codon use in the host used.

[0055] <Expression Vector> The expression vector of this disclosure has the nucleic acid of this disclosure as described above. The expression vector of this disclosure can express a region encoding a secreted fusion protein in a host cell. The expression vector may be any known vector, such as a plasmid vector or a viral vector. The known vector is not particularly limited as long as it can replicate in host cells such as bacteria such as Escherichia coli, fungi such as yeast, plant cells or animal cells, and examples include plasmid DNA and phage DNA. The known vector may be, for example, a commercially available expression vector for mammalian cells or a shuttle vector that can replicate between mammalian cells and bacteria such as Escherichia coli. Specifically, examples include pET vectors, pQE vectors, pCold vectors, and pUC19 vectors.

[0056] In particular, when expressing a target gene using Escherichia coli or the like as a host, it is especially preferable to use a plasmid vector. Furthermore, the expression vector preferably contains a transcription promoter, a transcription terminator, and a selection marker (a drug resistance gene, a gene that complements nutrient requirement mutations), and may also contain cis-elements such as enhancers, operators, and genes that control the promoter, if necessary. Here, suitable plasmid vectors include, for example, pET, pUC18, pUC19, pUC118, pUC119, pSC101, pBR322, pHSG298, pVC18, pVC19, pTrc99A, pMal-c2, pGEX2T, pTV118N, pTV119N, pTRP, etc. Methods for introducing plasmid vectors into E. coli include chemical transformation and electroporation, and these can be appropriately selected depending on the E. coli strain used and the size and properties of the plasmid vector to be introduced.

[0057] Furthermore, when using a host other than E. coli, plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, etc.), plasmids derived from Corynebacterium (e.g., pAM330, pHM1519, etc.), and plasmids derived from yeast (e.g., YEp13, YCp50, etc.) can be used. As phage vectors, while not limited to those mentioned, T7 phage display vectors and λ phage vectors can be used. As viral vectors, while not limited to those mentioned, animal viruses such as retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses, and Sendai viruses, as well as insect viruses such as baculoviruses, can be used. As cosmid vectors, while not limited to those mentioned, Lorist 6, Charomid 9-20, and Charomid 9-42 can be used. While not limited to phagemide vectors, examples of phagemide vectors that can be used include pSKAN, pBluescript, pBK, and pComb3H.

[0058] Furthermore, examples of expression vectors used with yeast as the host include pYES2 (Invitrogen), YEp13 (ATCC 37115), YEp24 (ATCC 37051), Ycp5O (ATCC 37419), pHS19, pHS15, etc. Specific examples of promoters for yeast include the PH05 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, and CUP1 promoter.

[0059] Furthermore, examples of expression vectors when using plant cells as hosts include plasmids such as Ti plasmid (Tumor inducing plasmid), pSPORT1, pT7 Blue-T vector, pIG121-Hm, and pBI121, or plant virus vectors such as tobacco mosaic virus, cauliflower mosaic virus, and geminivirus. Examples of promoters for plant cells include the cauliflower mosaic virus 35S promoter and the rubrose bisphosphate carboxylase small subunit promoter, and examples of terminators include the nopaline synthase gene terminator.

[0060] Furthermore, examples of expression vectors used with animal cells as hosts include plasmid vectors such as commercially available mammalian cell expression vectors like Promega's pCI vectors and pSI vectors, shuttle vectors that can replicate between mammalian cells and bacteria such as Escherichia coli, retroviral vectors (including oncoretroviral vectors, lentiviral vectors, and pseudotyped vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, Simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors. Examples of promoters for animal cells include the CMV promoter (CMV-IE promoter), SV40 initial promoter, RSV promoter, EF1α promoter, Ub promoter, and 5'LTR promoter.

[0061] <Transformed organisms> The transformed organisms of this disclosure have the nucleic acids of this disclosure as described above. The transformed organisms of this disclosure can also be prepared by introducing the expression vectors of this disclosure as described above into host cells. Host cells are not particularly limited, but may include bacteria such as Escherichia coli, Bacillus subtilis or Corynebacterium, fungi such as yeast, insect cells, animal cells (e.g., mammalian cells), and plant cells. The method for introducing the expression vectors into these host cells is not particularly limited, and methods such as calcium phosphate, electroporation, lipofection, particle gun, and PEG can be used as appropriate.

[0062] In particular, when using Escherichia coli as the host cell, for example, strains BL21, REL606, W3110, DH10B, BW25113, DH5α, MG1655, JM109, RV308, etc. can be used. Also, when using Bacillus subtilis as the host cell, for example, strains 168, CU1065, JH642, PY79, WB600, WB800, etc. can be used. Furthermore, when using Corynebacterium as the host cell, for example, Corynebacterium glutamicum ATCC13032, C. glutamicum R, C. glutamicum MB001, C. efficiens YS-314, C. Ammoniagenes strain ATCC6872, C. diphtheriae strain PW8, etc., can be used. These Escherichia coli, Bacillus subtilis, or Corynebacterium are usually prepared into competent cells suitable for gene transfer, and the expression vectors described above are introduced.

[0063] Furthermore, the host cells can also be yeasts belonging to genera such as Zygosaccharomyces, Saccharomyces, Pichia, and Candida. In addition, the host cells can also be filamentous fungi belonging to genera such as Aspergillus and Trichoderma. Furthermore, the host cells can also be animal cells such as human-derived cells, mouse-derived cells, hamster-derived cells, rat-derived cells, dog-derived cells, monkey-derived cells, and kangaroo-derived cells.

[0064] <Method for Producing Fusion Proteins> The method for producing fusion proteins according to the present disclosure includes the steps of culturing the transformants described above and recovering the fusion proteins according to the present disclosure from the culture medium in which the transformants were cultured. The method for producing fusion proteins according to the present disclosure prevents the fusion proteins secreted into the culture medium from being re-introduced into cells because they have a modified peptide that maintains heparan sulfate binding ability while reducing the cell permeability of the heparan sulfate binding peptide. Therefore, according to the method for producing fusion proteins according to the present disclosure, the target fusion protein can be produced in high yield.

[0065] The method for producing a fusion protein according to this disclosure can significantly increase the concentration of the fusion protein secreted into the culture medium compared to cases where the cell permeability of the heparan sulfate-binding peptide is not attenuated, for example, when the heparan sulfate-binding peptide itself is fused with the target protein. Here, "significant" means that there is a statistically significant difference. The method is not limited to known testing methods that can determine the presence or absence of significance. Examples include the t-test and multiple comparison tests. For example, a significant difference is determined when the significance level is less than 5%, 1%, 0.3%, 0.2%, or 0.1%. For example, according to the method for producing a fusion protein according to this disclosure, the concentration of the fusion protein in the culture medium can be 1 μg / mL or more, preferably 10 μg / mL or more, more preferably 100 μg / mL or more, and even more preferably 1000 μg / mL or more.

[0066] In the process of culturing the transformants, either a liquid medium or a solid medium may be used as the culture medium, but it is preferable to use a liquid medium in order to secrete and produce the target fusion protein. The composition of the medium can be appropriately selected depending on the type of transformant. If the transformant is a bacterial or fungal cell, the medium may contain one or more components selected from, for example, protein enzyme hydrolysates such as peptone and tryptone, biological extracts such as potato dextrose and yeast extract, amino acids such as glutamic acid or their salts, sugars such as glucose, glycerol, and sucrose, and inorganic salts such as sodium chloride, magnesium chloride, and potassium dihydrogen phosphate. Specific examples of mediums and compositions include LB medium, YPG medium, and PD medium. If the transformant is an animal cell, serum or plasma-containing medium, basic medium, or serum-free medium can be used as the culture medium. For example, commercially available mammalian cell basal media such as DMEM medium, IMDM medium, GMEM medium, HAM F10 medium, HAM F12 medium, and RPMI 1640 medium, to which serum or serum replacement solution has been added, may be used.

[0067] In the process of culturing the transformants, the culture temperature, the pH of the culture medium, and CO2 ２ Culture conditions such as concentration, culture time, and frequency of medium changes are not particularly limited and can be set as appropriate depending on the type of transformant, the secretion productivity of the target fusion protein, etc. For example, the culture time is not limited as long as it is the time required to obtain an antigen polypeptide containing a sufficient amount of sugar chains. For example, the culture time can be 1 hour or more, 2 hours or more, 4 hours or more, 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, or 1 week or more. The culture temperature can be 20-42°C, 25-40°C, 30-38°C, or 35-37°C. The pH of the medium can be pH 3-11, pH 4-10, pH 5-9, or pH 7-9.

[0068] Furthermore, if the transformant contains an expression vector, the process of culturing the transformant can be carried out in the presence of antibiotics or nutrients, depending on the drug resistance genes and nutritional requirement genes contained in the expression vector. Examples of antibiotics include ampicillin, kanamycin, tetracycline, and chloramphenicol.

[0069] Next, the method for producing the fusion protein of this disclosure involves a step of recovering the fusion protein of this disclosure from the culture medium in which the transformants were cultured. Specifically, the transformants and the culture medium are separated by methods such as centrifugation and filtration. The fusion protein contained in the culture medium can then be recovered and purified by applying chromatography such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reversed-phase column chromatography, and HPLC, as well as ammonium sulfate fractionation, ultrafiltration, and immunoadsorption.

[0070] In this case, if the fusion protein has the purification tags described above, the fusion protein can be easily recovered from the culture medium by affinity chromatography corresponding to the purification tag. Examples include His tag, Flag tag, Spot tag, C tag, Strip tag, GST tag, MBP tag, etc. If the fusion protein has an HIS tag, immobilized metal affinity chromatography is used. If the fusion protein has a Flag tag, affinity chromatography with an immobilized Flag tag antibody is used. If the fusion protein has a Spot tag, affinity purification can be performed using a VHH antibody specific to the Spot tag. If the fusion protein has a GST tag, purification can be performed using an anti-GST antibody or glutathione. If the fusion protein has an MBP tag, purification can be performed by affinity chromatography using an anti-MBP antibody or amylose resin.

[0071] Incidentally, the fusion protein according to this disclosure can also be produced based on its amino acid sequence by peptide synthesis methods such as liquid-phase synthesis and solid-phase synthesis, or by peptide synthesis using an automated peptide synthesizer. In peptide synthesis, for example, amino acids are prepared in which functional groups other than the α-amino group and α-carboxyl group are protected for each amino acid, and a peptide bond formation reaction is carried out between the α-amino group and α-carboxyl group of each amino acid. Typically, the carboxyl group of the amino acid residue located at the C-terminus of the peptide is attached to the solid phase via an appropriate spacer or linker. The protecting group at the amino terminus of the dipeptide thus obtained is selectively removed, and a peptide bond is formed with the α-carboxyl group of the next amino acid. By performing this operation sequentially, peptides with protected side groups are produced, and finally, all protecting groups are removed and separated from the solid phase. The fusion protein according to this disclosure can be synthesized by the above method.

[0072] The methods for producing oligonucleotides according to this disclosure will be described in more detail below with reference to examples, but the technical scope of this disclosure is not limited to the following examples.

[0073] [Experiment 1] In Experiment 1, a secreted fusion protein was constructed using a single-domain antibody (VHH), which is an agonist for human fibroblast growth factor receptor 2 (FGFR2), as the target protein. The VHH used in this example is disclosed as VHH Clone #1 in International Publication No. 2022 / 270518. The amino acid sequence of the VHH is shown in Sequence ID No. 38.

[0074] Furthermore, the secreted fusion protein in this example has reduced cell permeability compared to the original heparan sulfate-binding peptide, and possesses a modified peptide (KTRYKARRA (SEQ ID NO: 1), denoted as HSBP1) that maintains its ability to bind to heparan sulfate. In addition, the secreted fusion protein in this example has a pair of the above VHH sequences, and a linker sequence (GSEGKSSGSGSESKST (SEQ ID NO: 39), denoted as Linker) between these VHH sequences. Moreover, the secreted fusion protein in this example has a tag sequence for purification at its C-terminus (GGGGSEPEA (SEQ ID NO: 40), denoted as tag).

[0075] In this experiment, we designed secreted fusion proteins for the comparative example that did not contain the modified peptide, and secreted fusion proteins for several examples in which the position of the modified peptide was changed. The compositions of the secreted fusion proteins for the examples and comparative examples are summarized in Table 2.

[0076]

[0077] 1. Preparation of Expression Vectors In this example, an expression vector was prepared by inserting a Lac promoter sequence (SEQ ID NO: 41), a sequence encoding a pelB secretion signal sequence (SEQ ID NO: 42), an AscI restriction enzyme site, a sequence encoding the above tag sequence (SEQ ID NO: 43), and a stop codon into the multi-cloning site (MCS) of the cloning vector pTZ19R.

[0078] 2. Insertion of Insert Sequence into Expression Vector The expression vector prepared in 1. above was treated with the restriction enzyme AscI, and cleaved at the AscI restriction enzyme site in the MCS of the expression vector. Then, an oligonucleotide with a base sequence artificially synthesized based on the amino acid sequence of the secreted fusion protein with the configuration shown in Table 2 was inserted into the cleavage site using NEBuilder HiFi DNA Assemblely Master Mix (NEB). In addition, since NEBuilder HiFi DNA Assemblely Master Mix was used for the base sequence encoding the secreted fusion protein, CAACCAGccatggCA (SEQ ID NO: 44, part of the pelB secretion signal sequence) was added to the 5' side and GGaGGcGGtGGgAGt (SEQ ID NO: 45, part of the tag sequence) was added to the 3' side.

[0079] 3. Transformation The expression vector expressing the secreted fusion protein prepared in 2. above was introduced into Escherichia coli (ElectroMAX DH12S Cells (Thermo Fisher Scientific)) by electroporation. Subsequently, the cells were seeded onto an agar medium containing ampicillin (final concentration 100 μg / mL), and the formation of transformed E. coli colonies was confirmed.

[0080] 4. Culture First, as a pre-culture, a culture medium was prepared by adding 2×YT medium, ampicillin (final concentration 100 μg / mL), and glucose (final concentration 1% by mass) to a test tube. Then, the transformed Escherichia coli obtained in 3. above was inoculated into this medium and cultured with shaking at 30°C for 16 to 20 hours. Then, as the main culture, 2×YT medium, ampicillin (final concentration 100 μg / mL), glucose (final concentration 0.05% by mass), IPTG (final concentration 1 mM), and pre-culture solution (1 / 100th volume) were added to an Erlenmeyer flask and cultured with shaking at 30°C for 18 to 20 hours.

[0081] 5. Recovery of the culture supernatant (solution containing fusion protein) The culture solution obtained in step 4 above was transferred to a centrifuge tube and centrifuged at 10,000 G, 4°C, for 10 minutes. The obtained supernatant was filtered through a sterile filter with a pore size of 0.22 μm and transferred to a new container.

[0082] 6. SDS-PAGE (Sodium dodecyl sulfate)-polyacrylamide gel electrophoresis was performed using 10 μL of Western blotting supernatant. Subsequently, the proteins separated from the gel were transferred to a membrane and blocked with PBS / 5% by mass skim milk solution. Then, they were reacted with CaptureSelect HRP Anti-C-tag Conjugate (Thermo Fisher Scientific) diluted 1,000-fold with TBS-T (TBS / 0.05% by mass tween20) solution and incubated at room temperature for 1 hour. After washing three times with TBS-T solution, they were reacted with the chemiluminescent reagent Amersham ECL (Cytiva), and after 2 minutes at room temperature, images were taken using a chemiluminescence detector. Furthermore, the culture supernatant was diluted to 1 / 10 and Western blotting was performed in the same manner, and images were taken using a chemiluminescence detector.

[0083] 7. Results The results of Western blotting for the secreted fusion proteins of Examples 1 to 4 and Comparative Example 1 are shown in Figure 2. It was revealed that the secreted fusion proteins of Examples 1 to 4, despite possessing HSBP1, a partial peptide of the heparan sulfate-binding peptide, were capable of extracellular secretion by transformed E. coli. This result indicates that the HSBP1 used in these examples has reduced cell permeability compared to the original heparan sulfate-binding peptide.

[0084] Furthermore, as shown in Figure 2, bands originating from the linker sequence or partially cleaved proteins at HSBP1 were observed in the lanes of the secreted fusion proteins in Examples 2 and 3. This suggests that it is preferable for HSBP1 to be located at the N-terminus or C-terminus of the secreted fusion protein. In particular, it was found that the secreted fusion protein of Example 1, which has HSBP1 at the N-terminus, was produced in a relatively high volume compared to the other secreted fusion proteins.

[0085] [Experiment 2] In this experiment, secreted fusion proteins were constructed using HSBP2 (TRRQRT (SEQ ID NO: 2)), HSBP3 (SRPRRP (SEQ ID NO: 3)), and HSBP4 (NRRMKWKK (SEQ ID NO: 30)) instead of HSBP1 (KTRYKARRA (SEQ ID NO: 1)). In this example, the secreted fusion proteins of Examples 5 to 10 were prepared using the same procedure as in Experiment 1 (1 to 5 above). The composition of the secreted fusion protein of Example 1 and the secreted fusion proteins of Examples 5 to 10 constructed in this experiment are summarized in Table 3.

[0086]

[0087] Figure 3 shows the results of Western blotting of the secreted fusion proteins of Examples 1 and 5-10 in the same manner as in Experiment 1, Section 6 described above. It was revealed that the secreted fusion proteins of Examples 5-10, despite containing one of the heparan sulfate-binding peptide partial peptides HSBP2-HSBP4, similar to the secreted fusion protein of Example 1, can be secreted and produced extracellularly by transformed E. coli. This result indicates that HSBP2-HSBP4 used in this example have reduced cell permeability compared to the original heparan sulfate-binding peptide.

[0088] Furthermore, as shown in Figure 3, it was found that when HSBP2 and HSBP3, which have a lower proportion of basic amino acids (lysine, arginine, and histidine), were used, the secretion production was higher compared to when HSBP1 or HSBP4 were used.

[0089] [Experiment 3] In this experiment, the secreted fusion proteins of Example 5 and Example 6 were purified after performing steps 1 to 5 of Experiment 1 described above, and before Western blotting (step 6). Specifically, the secreted fusion proteins were purified from the culture supernatant using CaptureSelect C-tagXL Pre-packed Column (Thermo Fisher Scientific) with the tag portion. The specific conditions were as follows: - Equilibrium: TBS solution, 5 column volume (CV), 2 mL / min - Application of culture supernatant: 1 mL / min - Washing: 10 CV, 2 mL / min - Elution: TBS / 2M MgCl ２ Solution, 10 CV, 1 mL / min, fractions collected and buffer replaced in 1 CV increments: Prepacked Disposable PD-10 Columns (Cytiva) with TBS / 2M MgCl ２ Replace the buffer from the solution with PBS solution.

[0090] As described above, the secreted fusion proteins of Example 5 and Example 6, purified as shown above, were subjected to Western blotting in the same manner as in Experiment 1, Section 6, as shown in Figure 4. Figure 4 also shows the secreted fusion protein of Comparative Example 1, which was similarly purified. As shown in Figure 4, it was found that when using HSBP2 with a low proportion of basic amino acids, it was possible to produce a high amount of secreted fusion protein, comparable to that of Comparative Example 1, which does not contain heparan sulfate-binding peptides. This result demonstrates that by reducing the proportion of basic amino acids in the heparan sulfate-binding peptide, a sufficient amount of secreted fusion protein can be produced even after purification.

[0091] [Experiment 4] In this experiment, the binding ability of the secreted fusion protein of Example 5 and the secreted fusion protein of Example 6 to heparan sulfate was evaluated. First, biotin-labeled sodium heparan sulfate (PG Research) and 4 μg / mL in 1x PBS / 0.1% Triton X-100 (hereinafter referred to as PBS / T) were dispensed into streptavidin-coated ELISA plates (ThermoFisher SCIENTIFIC) to a concentration of 100 μL / well, and the plates were left to stand at 37°C for 1 hour. After washing three times with PBS / T to a concentration of 300 μL / well, the secreted fusion proteins of Example 5, Example 6, and Comparative Example 1, diluted with PBS / T to 30.0 μg / mL, 7.5 μg / mL, 1.9 μg / mL, and 0.5 μg / mL, were dispensed in 100 μL / well volumes and allowed to stand at 37°C for 1 hour. After washing twice with PBS / T to a concentration of 300 μL / well, CaptureSelect HRP Anti-C-tag Conjugate (manufactured by ThermoFisher SCIENTIFIC), diluted 1,000 times with PBS / T, was dispensed in 100 μL / well volumes and allowed to stand at 37°C for 1 hour. After washing three times with PBS / T to a volume of 300 μL / well, TMB / E Solution (Merck Millipore) was dispensed to a volume of 100 μL / well and allowed to stand at room temperature for 5 minutes. 0.3 M sulfuric acid was then dispensed to a volume of 100 μL / well, and after stopping the reaction, the absorbance was measured using a plate reader (absorbance: 450 nm).

[0092] The results measured with a plate reader are shown in Figure 5. As shown in Figure 5, it was revealed that the purified secreted fusion protein containing HSBP2 possessed the ability to bind to heparan sulfate. Combining the results of Experiments 2 and 3 described above with the results of this experiment, it was revealed that the modified peptide HSBP2 with a lower proportion of basic amino acids had reduced cell permeability, enabling the secretion and production of the target protein while maintaining its ability to bind to heparan sulfate. Furthermore, it was revealed that the secreted fusion protein with HSBP2 at the N-terminus (Example 5) had superior binding ability to heparan sulfate compared to the secreted fusion protein with HSBP2 at the C-terminus (Example 6). This suggests that the negative charge of the C-terminal tag sequence may have acted to cancel the positive charge of HSBP2.

[0093] [Experiment 5] In this experiment, the secreted fusion protein of Example 5 and the secreted fusion protein of Example 6, which were constructed in Experiment 2, were modified as follows.

[0094] In this experiment, the secreted fusion protein of Example 11 was constructed by introducing a linker (GGGGS (SEQ ID NO: 46), denoted as Linker2) between the modified peptide HSBP2 and the target protein in the secreted fusion protein of Example 5. Furthermore, in this experiment, the secreted fusion protein of Example 12 was constructed by introducing a cleavage-promoting sequence A (alanine, denoted as CPS1 (Clevagage Promotion Sequence 1)) between the pelB secretion signal sequence and the modified peptide HSBP2 in the secreted fusion protein of Example 11. In addition, in this experiment, the secreted fusion protein of Example 13 was constructed by introducing a cleavage-promoting sequence AQ (alanine-glutamine, denoted as CPS2 (Clevagage Promotion Sequence 2)) between the pelB secretion signal sequence and the modified peptide HSBP2 in the secreted fusion protein of Example 11. Furthermore, in this experiment, we constructed the secreted fusion protein of Example 14 by introducing Linker2 between the target protein and HSBP2 in the secreted fusion protein of Example 6, and introducing a different tag sequence (SEPEA (SEQ ID NO: 47), denoted as tag2) in place of tag at the C-terminus.

[0095] Table 4 summarizes the composition of the secreted fusion protein of Example 5, the secreted fusion protein of Example 6, and the secreted fusion proteins of Examples 11 to 14 constructed in this experiment.

[0096]

[0097] In this example, the secreted fusion proteins of Examples 11 to 13 were prepared using the same procedure as in Experiment 1 described above (steps 1 to 5 above).

[0098] For the secreted fusion protein of Example 14, in the procedure of Experiment 1 described above, during "1. Preparation of Expression Vector," an expression vector was used in which the Lac promoter sequence (SEQ ID NO: 41), the pelB secretion signal sequence (SEQ ID NO: 42), the AscI restriction enzyme site, the tag2 encoding sequence (AGtGAACCAGAGGCG, SEQ ID NO: 48), and a stop codon were inserted into the multi-cloning site (MCS) of the cloning vector pTZ19R. Furthermore, for the secreted fusion protein of Example 14, in the procedure of Experiment 1 described above, during "2. Insertion of Insert Sequence into Expression Vector," CAACCAGccatggCA (SEQ ID NO: 44, part of the pelB secretion signal sequence) was added to the nucleotide sequence encoding the secreted fusion protein at the 5' end and the tag2 encoding sequence (SEQ ID NO: 48) at the 3' end, in order to use NEBuilder HiFi DNA Assemblely Master Mix.

[0099] Figure 6 shows the results of Western blotting of the secreted fusion protein of Example 5, the secreted fusion protein of Example 6, and the secreted fusion proteins of Examples 11 to 14 constructed in this experiment, in the same manner as in Experiment 1, Section 6 described above. Furthermore, the secreted fusion protein of Example 5, the secreted fusion protein of Example 6, the secreted fusion protein of Example 13, and the secreted fusion protein of Example 14 were purified using the method described in Experiment 3 described above. Subsequently, the binding ability of the secreted fusion protein of Example 5, the secreted fusion protein of Example 6, the secreted fusion protein of Example 13, and the secreted fusion protein of Example 14 to heparan sulfate was evaluated using the method described in Experiment 4 described above. Figure 7 shows the results of plate reader measurements for the secreted fusion protein of Example 5 and the secreted fusion protein of Example 13, and Figure 8 shows the results of plate reader measurements for the secreted fusion protein of Example 6 and the secreted fusion protein of Example 14.

[0100] As shown in Figure 6, it was revealed that production could be increased by introducing a linker between HSBP2 and the target protein in the secreted fusion protein of Example 5 (secreted fusion proteins of Examples 11 to 13), and that production could be further increased by introducing a cleavage-promoting sequence between the secretory signal sequence and HSBP2 (secreted fusion proteins of Examples 12 and 13). Furthermore, as shown in Figure 7, it was revealed that the ability to bind to heparan sulfate was maintained even when a linker was introduced between HSBP2 and the target protein, and when a cleavage-promoting sequence was introduced between the secretory signal sequence and HSBP2.

[0101] Furthermore, as shown in Figure 6, it was revealed that the secreted fusion protein of Example 14, in which linker 2 was introduced between the target protein and HSBP2 in the secreted fusion protein of Example 6 and tag2 was used at the C-terminus, could be produced at a similarly high rate as the secreted fusion protein of Example 6. In addition, as shown in Figure 8, it was revealed that the secreted fusion protein of Example 14 had improved binding ability to heparan sulfate compared to the secreted fusion protein of Example 6. This can be attributed to the fact that the tag at the C-terminus of the secreted fusion protein of Example 6 contains four glycine (flexible linker) molecules, while the tag2 at the C-terminus of the secreted fusion protein of Example 14 contains one serine molecule. In other words, it was inferred that the tag at the C-terminus of the secreted fusion protein of Example 6 may have interfered with the binding of HSBP2 to heparan sulfate. In contrast, in the secreted fusion protein of Example 14, the C-terminal tag2 did not contain a flexible linker, and therefore it was inferred that the C-terminal tag2 did not interfere with the binding of HSBP2 to heparan sulfate.

[0102] [Experiment 6] In this experiment, in order to compare it with the secreted fusion protein of Example 14 constructed in Experiment 5, a secreted fusion protein of Comparative Example 2 was designed using a known heparan sulfate-binding peptide instead of HSBP2. Specifically, the secreted fusion protein of Comparative Example 2 uses HTLV-II Rex (TRRQRTRRARRNR (SEQ ID NO: 7), see Table 1) instead of HSBP2 in the secreted fusion protein of Example 14.

[0103] The secreted fusion protein of Example 14 and the secreted fusion protein of Comparative Example 2 were prepared using the same procedure as in Experiment 5 described above, and Western blotting was performed in the same manner as in Experiment 1, Section 6 described above, except that the culture supernatant was diluted to 1 / 50. The results of the Western blotting are shown in Figure 9. As shown in Figure 9, extracellular secretion production of the secreted fusion protein of Comparative Example 2 could not be detected. From these results, it became clear that secreted fusion proteins can be secreted extracellularly by attenuating the cell permeability of known heparan sulfate-binding peptides.

[0104] 1...Fusion protein 2A, 2B...Interaction site 3...Modified peptide 4...Receptor 5...Molecule containing heparan sulfate 6...Other protein

[0105] The disclosure of Japanese Patent Application No. 2025-17698, filed on 5 February 2025, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually noted to be incorporated by reference.

Claims

1. A secretory fusion protein comprising a secretory signal sequence, a modified peptide in which the cell permeability of the heparan sulfate-binding peptide is attenuated while the heparan sulfate-binding ability is maintained, and the target protein.

2. The secreted fusion protein according to claim 1, wherein the modified peptide is the heparan sulfate binding domain in the heparan sulfate binding peptide.

3. The secretory fusion protein according to claim 1, wherein the modified peptide has 75% or less basic amino acid residues among all amino acid residues constituting the modified peptide.

4. The secretory fusion protein according to claim 1, wherein the modified peptide has 50% or less basic amino acid residues among all amino acid residues constituting the modified peptide.

5. The modified peptide is a single amino acid sequence selected from the group consisting of (a) to (d) below, according to claim 1, for the secreted fusion protein: (a) KTRYKARRA (SEQ ID NO: 1) (b) TRRQRT (SEQ ID NO: 2) (c) SRPRRP (SEQ ID NO: 3) (d) NRRMKWKK (SEQ ID NO: 30) 6. The secreted fusion protein according to claim 1, wherein the target protein interacts with proteins present on the cell surface.

7. The secreted fusion protein according to claim 6, wherein the protein present on the cell surface is a receptor, and the target protein is an agonist for the receptor.

8. The secreted fusion protein according to claim 7, wherein the receptor is a receptor with which a growth factor interacts.

9. The growth factors include EGF, GM-CSF, M-CSF, IL-6, PDGF-AA, PDGF-BB, IL-2, FGF-2, IL-3, IL-7, TGF-β1, FLt3-Ligand, IL-4, TPO, IL. -5, IL-15, IL-21, IL-18, IFN-γ, SCF, VEGF165, BMP-4, TNF-alpha, Noggin, R-Spondin, Wnt-3a, FGF-10, KGF, Activin The secreted fusion protein according to claim 8, which is a growth factor selected from the group consisting of A, IGF-1, NRG1, Jaggered1, VEGF121, HGF, BMP-2, Shh, FGF-8, BDNF, GDNF, Artemin, Beta-NGF, CDNF, CNTF, GMF-beta, MANF, Midkine, NT-3, NT-4, NGF, Pleiotropin, TrkA, TrkB, and TrkC.

10. The secreted fusion protein according to claim 1, wherein the target protein has a pair of interaction sites with a protein present on the cell surface.

11. The secreted fusion protein according to claim 10, wherein the interaction site is a small molecule antibody selected from the group consisting of a single-chain antibody (scFv) and a heavy chain antibody variable region (VHH) antibody that specifically binds to the protein.

12. The secreted fusion protein according to claim 1, having a linker between the modified peptide and the target protein.

13. The secreted fusion protein according to claim 12, wherein the linker is a flexible linker.

14. The secreted fusion protein according to claim 13, wherein the flexible linker comprises an amino acid sequence of 5 amino acid residues or less, including 2 or more glycine residues.

15. The secretory fusion protein according to claim 1, having a cleavage-promoting sequence between the secretory signal sequence and the modified peptide or the target protein.

16. The secreted fusion protein according to claim 15, wherein the cleavage-promoting sequence is A or AQ.

17. The secreted fusion protein according to claim 1, further having a purification tag sequence at its C-terminus.

18. The secreted fusion protein according to claim 17, wherein the modified peptide is linked to the tag sequence directly or via a linker.

19. The secreted fusion protein according to claim 18, wherein the linker has an amino acid sequence consisting of amino acids other than glycine.

20. A fusion protein obtained by cleaving a region containing a secretory signal sequence from a secretory fusion protein according to any one of claims 1 to 19.

21. A nucleic acid encoding a secretory fusion protein according to any one of claims 1 to 19.

22. An expression vector having the nucleic acid described in claim 21.

23. A transformant having the nucleic acid described in claim 21.

24. A method for producing a fusion protein, comprising the steps of: culturing the transformant described in claim 23; and recovering the fusion protein described in claim 20 from the culture medium in which the transformant was cultured.

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