Zinc-dependent metalloproteinases that can degrade abeta42

Abstract

The present invention provides a zinc-dependent metalloprotease that can degrade Aβ42. The protease of the present invention is: (a) a protein having an amino acid sequence selected from any one of SEQ ID NOs: 1 to 6; or (b) a protein derived from (a) with substitution, deletion or addition of one or several amino acids in the amino acid sequence of (a) and having amyloid beta (Aβ) degrading activity. The protease of the present invention is a completely de novo designed zinc-dependent metalloprotease that recognizes target polypeptides by a structurally pre-organized active center to independently achieve active catalytic degradation by a single molecule.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a zinc-dependent metalloproteinase that can degrade Aβ42. Background Technology

[0002] The abnormal aggregation and deposition of amyloid-β (Aβ) is a core pathological feature of Alzheimer's disease. Among these, the Aβ42 subtype, due to its tendency to form highly neurotoxic soluble oligomers and insoluble fibrillary plaques, is considered the primary driver of the disease's progression. Therefore, effectively degrading and clearing Aβ42 aggregates in the central nervous system has become a top priority in current macromolecular drug development. Currently, targeted clearance strategies mainly rely on anti-Aβ monoclonal antibodies. However, these drugs rely on a stoichiometric passive binding mechanism to label Aβ, which, limited by the extremely low permeability of the blood-brain barrier, typically requires very high systemic doses to achieve effective concentrations in the brain. This passive binding mode not only has limited therapeutic efficiency, but its high-dose systemic exposure also poses serious safety risks.

[0003] To overcome the limitations of passive binding by antibody drugs, catalytic enzyme therapy that directly hydrolyzes Aβ has emerged as a promising alternative intervention. Based on the cyclical nature of enzymes, a single protease molecule can continuously degrade multiple Aβ substrates, theoretically enabling long-term clearance of pathological peptides with extremely low dosages. However, existing natural proteases (such as endogenous enkephalinase or insulin-degrading enzymes) have evolved broad substrate diversity and completely lack absolute selectivity for pathogenic Aβ sequences. Introducing such natural enzymes directly or through simple modification into the central nervous system to amplify their Aβ clearance capacity can easily trigger unintended hydrolysis of other key physiological target proteins, leading to uncontrollable and severe off-target toxicity risks.

[0004] Therefore, current technologies lack a safe removal tool that can efficiently catalyze the cleavage of Aβ while strictly avoiding off-target toxicity. How to obtain targeted metalloproteinases with both high catalytic activity and extremely high Aβ substrate selectivity to achieve low-dose, active, and safe degradation of pathogenic peptides is a core technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] This invention discloses a zinc-dependent metalloproteinase designed entirely de novo to target amyloid-β (Aβ) and its application as a drug lead molecule.

[0006] This invention aims to provide a completely de novo-designed zinc-dependent metalloproteinase that independently and actively catalyzes the degradation of target peptides by recognizing them through a pre-organized active site. The core mechanism for achieving precise cleavage is selected from metal-coordination center interactions, substrate-binding interface interactions, and transition state stabilization network interactions. For metal-coordination center-mediated catalytic degradation, zinc ions interact with precisely arranged histidine and aspartic acid residues on the protease backbone, forming stable sub-angstrom tetrahedral coordination, thereby effectively activating water molecules to initiate catalysis. For substrate-binding interface-mediated catalytic degradation, designed pocket amino acids interact with the disordered regions of the amyloid-β (Aβ) substrate, forming tight bindings with highly complementary shapes, thus endowing the enzyme with initial substrate selectivity. For transition state stabilization network-mediated catalytic degradation, auxiliary catalytic residues around the active site interact with transient intermediates during substrate peptide bond cleavage, thereby completing the cleavage of the target peptide.

[0007] Therefore, in a first aspect, the present invention provides a protease, said protease being: (a) Having an amino acid sequence selected from any one of SEQ ID NO: 1 to 6; or (b) A protein derived from (a) whose amino acid sequence in (a) has been substituted, deleted or added with one or more amino acids and has the activity of degrading amyloid β (Aβ) (especially Aβ42).

[0008] In one embodiment, the protease has the amino acid sequence shown in SEQ ID NO: 1.

[0009] In one embodiment, the protease has the amino acid sequence shown in SEQ ID NO: 2.

[0010] In one embodiment, the protease has the amino acid sequence shown in SEQ ID NO: 3.

[0011] In one embodiment, the protease has the amino acid sequence shown in SEQ ID NO: 4.

[0012] In one embodiment, the protease has the amino acid sequence shown in SEQ ID NO: 5.

[0013] In one embodiment, the protease has the amino acid sequence shown in SEQ ID NO: 6.

[0014] In this invention, the protease is a zinc-dependent metalloproteinase capable of degrading Aβ (especially Aβ42).

[0015] In a second aspect, the present invention provides a polynucleotide that encodes the protease of the first aspect.

[0016] In a third aspect, the present invention provides a recombinant vector comprising the polynucleotide of the second aspect.

[0017] In some embodiments, the recombinant vector may be an expression vector.

[0018] In a fourth aspect, the present invention provides a recombinant cell comprising a protease of the first aspect, a polynucleotide of the second aspect, or a recombinant vector of the third aspect.

[0019] In a fifth aspect, the present invention provides a composition comprising a protease in the first aspect, a polynucleotide in the second aspect, a recombinant vector in the third aspect, or a recombinant cell in the fourth aspect.

[0020] In a sixth aspect, the present invention provides the use of the protease of the first aspect, the polynucleotide of the second aspect, the recombinant vector of the third aspect, the recombinant cell of the fourth aspect, or the composition of the fifth aspect in the preparation of amyloid β (especially Aβ42) degrading agents or scavengers.

[0021] In a seventh aspect, the present invention provides the use of the protease of the first aspect, the polynucleotide of the second aspect, the recombinant vector of the third aspect, the recombinant cell of the fourth aspect, or the composition of the fifth aspect in the preparation of a medicament for the prevention or treatment of diseases caused by abnormal aggregation of amyloid β (especially Aβ42). In some embodiments, the disease is Alzheimer's disease. Attached Figure Description

[0022] Figure 1 The results of enzyme activity monitoring using fluorescence resonance energy transfer (FRET) substrates are shown.

[0023] Figure 2 This is the mass spectrum of the hydrolysis product of PP507-S1 on Aβ42.

[0024] Figure 3 This is the mass spectrum of the hydrolysis product of PP532-S1 on Aβ42.

[0025] Figure 4 This is the mass spectrum of the hydrolysis product of DP622-S2 on Aβ42.

[0026] Figure 5 This is the mass spectrum of the hydrolysis product of Aβ42 by DP221-S3.

[0027] Figure 6 This is the mass spectrum of the hydrolysis product of Aβ42 by OP609-S2.

[0028] Figure 7 This is the mass spectrum of the hydrolysis product of Aβ42 by OP669-S2.

[0029] Figure 8 Cryo-electron microscopy density images of zinc-dependent metalloproteinases PP509-S1, DP622-S2, and DP221-S3 with their substrates.

[0030] Figure 9 Local and global resolution images of zinc-dependent metalloproteinases PP509-S1, DP622-S2, and DP221-S3 with their substrates obtained by cryo-electron microscopy. Detailed Implementation

[0031] The present invention will be described in detail below by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] Example 1: Expression and purification of zinc-dependent metalloproteinase protein The gene encoding a zinc-dependent metalloproteinase was cloned into a pET-28a(+) *E. coli* expression vector with an N-terminus of an 8×His tag and a thrombin cleavage site (MGHHHHHHHHSSGLEVLFQGPGGT (SEQ ID NO: 12)). The gene synthesis and vector construction were performed by Tianjin Jiutian Gene Technology Co., Ltd. The cloned plasmid was transformed into *E. coli* BL21(DE3) (EC1002, WEIDIBio) and cultured in 0.5 L LB broth at 37°C and 220 RPM until the OD600 reached 0.6–0.8. The culture was then cooled to 20°C, and IPTG (206-703-0, MACKLIN) was added to a final concentration of 0.5 mM. The culture was incubated overnight at 20°C and 220 RPM. Cells were collected by centrifugation at 5000g for 5 minutes and resuspended in 40 ml protein buffer (25 mM Tris-HCl (pH 7.4) + 150 mM NaCl). The cells were then sonicated for 15 minutes. Whole-cell lysates were separated by centrifugation at 12000 RPM for 30 minutes, and the target protein was purified from the supernatant using Ni-NTA resin. The target protein solution was mixed with HRV 3C protease (purchased from Shanghai Yisheng Biotechnology Co., Ltd.), with a mass ratio of 50:1. The mixture was dialyzed overnight in 1 L protein buffer to cleave the N-terminal His tag. The dialyzed protein solution was centrifuged at 4000g for 10 minutes, and the supernatant was collected. Undigested protein was removed from the supernatant using Ni-NTA resin. 1 mM EDTA was added to the treated protein solution, and after concentration, the target protein was further purified by molecular sieve chromatography using a Superdex 200 Increase 10 / 300 GL column (28990944, Cytiva) in protein buffer. The protein concentration of the protein solution obtained by molecular sieve chromatography was measured using a NanoPhotometer N50 (implen), and the molar concentration of the protein was calculated from the protein molecular weight.

[0035] Example 2: Enzyme activity curves of zinc-dependent metalloproteinases cleaving fluorescent substrates In this embodiment, the kinetic tests used the chimeric fluorescence resonance energy transfer (FRET) substrates mTurquoise2-Aβ-mVenus16 (TV-S1, TV-S2, TV-S3, TV-S4), where the targeted Aβ core sequence served as a linker that could be specifically cleaved. The chimeric FRET substrates were used solely as an observational tool to determine hydrolytic activity. The FRET substrates (TV-S1, TV-S2, TV-S3, TV-S4) were prepared using the expression and purification methods described in Example 1.

[0036] To accurately convert the raw fluorescence signal into substrate conversion, a standard curve was first established. The FRET substrate was hydrolyzed using a highly active, pre-designed enzyme (e.g., OP609-S2) in a reaction buffer containing 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 10 times the enzyme molar concentration of ZnSO4. The complete hydrolysate was then mixed with unreacted intact substrate at different molar ratios (0:15, 1:14, 2:13, 3:12, 4:11, 5:10, 6:9, 7:8, 8:7, 9:6, 10:5, 11:4, 12:3, 13:2, 14:1, 15:0). Using the Varioskan™ LUX multi-functional microplate reader from Thermo Fisher Scientific, the ratio of emission wavelengths of 527 nm (acceptor) to 474 nm (donor) for each standard sample at an excitation light of 434 nm was recorded (R = I527 / I474), establishing a linear correlation between the fluorescence ratio and the degree of substrate hydrolysis.

[0037] Enzymatic kinetics were performed in black flat-bottomed 96-well microplates to minimize interwell background scattering. Purified zinc-dependent metalloproteinases were adjusted to specific concentrations (PP507-S1: 2.5 μM; PP532-S1: 2.5 μM; DP622-S2: 2.5 μM; DP221-S3: 5 μM; OP609-S2: 0.2 μM; OP669-S2: 2.5 μM) and co-incubated with FRET substrates in a buffer containing 25 mM Tris HCl (pH 7.4) and 150 mM NaCl. A ten-molar excess of ZnSO4 was added to the reaction system to ensure optimal pre-organization of the metalloproteinase active sites and tight binding to the peptide substrates. FRET substrate concentrations ranged from 20 μM to 0.156 μM.

[0038] Kinetic fluorescence monitoring was performed continuously at 25°C, with an excitation wavelength of 434 nm and dual emission wavelengths of 474 nm (donor) and 527 nm (acceptor). During data analysis, the initial reaction rate was derived from the linear phase data of product formation. Subsequently, nonlinear regression was performed using GraphPad Prism 9.0 software to fit the initial reaction rate and corresponding substrate concentration to the Michaelis-Menten equation, thereby accurately calculating and recording the enzyme's catalytic constant (kcat), Michaelis constant (Km), and catalytic efficiency (kcat / Km). All kinetic measurements were independently repeated three times to ensure the rigor of the experimental procedures and data reproducibility.

[0039] The enzyme activities of PP507-S1 and PP532-S1 were detected by FRET substrate TV-S1, the enzyme activities of DP221-S3 were detected by FRET substrate TV-S3, the enzyme activities of DP622-S2 and OP609-S2 were detected by FRET substrate TV-S2, and the enzyme activities of OP669-S2 were detected by FRET substrate TV-S4.

[0040] Figure 1 The results of enzyme activity monitoring using fluorescence resonance energy transfer (FRET) substrates are shown. It can be seen that the zinc-dependent metalloproteinases designed in this invention exhibit highly efficient catalytic cleavage activity against the corresponding target peptide substrates. Specifically, the catalytic efficiency of PP507-S1 is 54.67 M. -1 s -1 The catalytic efficiency of PP532-S1 is 48.09 M. -1 s -1 The catalytic efficiency of DP221-S3 is 9.50 M. -1 s -1 The catalytic efficiency of DP622-S2 is 325.26 M. -1 s -1 The catalytic efficiency of OP609-S2 is 3045.14 M. -1 s -1 The catalytic efficiency of OP669-S2 is 452.49 M. -1 s -1 This technical effect fully demonstrates that the recombinant protease described in this invention can degrade the target sequence and has the potential to be used as an Aβ42 peptide degrader.

[0041] Example 3: Mass spectrum of Aβ42 degradation by zinc-dependent metalloproteinases First, the Aβ42 monomeric peptide substrate was prepared. Aβ42 peptide powder (purchased from Shanghai Chutai Biotechnology Co., Ltd.) was first dissolved in hexafluoroisopropanol (HFIP, purchased from Aladdin) to prepare a 1 mM solution to thoroughly disrupt and depolymerize any pre-existing peptide aggregates. This solution was incubated at room temperature for 1 hour to ensure sufficient and complete monomerization, and then placed in a fume hood overnight to allow the HFIP to evaporate naturally. To completely remove any residual organic solvent, the resulting substrate peptide film was further dried for 1 hour using a Christ CT 02-50 vacuum centrifuge. Finally, the completely dried peptide film was directly dissolved in dimethyl sulfoxide (DMSO) to obtain a high-purity Aβ42 monomer stock solution.

[0042] Subsequently, an experiment was conducted to determine the hydrolytic activity of zinc-dependent metalloproteinases. The reaction used the monomer Aβ42 prepared above as the hydrolysis substrate. In the test system, the Aβ42 monomer substrate to a final concentration of 20 μM was thoroughly mixed with the zinc-dependent metalloproteinase to a final concentration of 10 μM. The catalytic reaction was incubated overnight in a buffer solution containing 25 mM Tris HCl (pH 7.4) and 150 mM NaCl. To ensure precise coordination of the metal ion at the active site, ZnSO4 at an equimolar concentration (10 μM) to the protein was also added to the buffer solution. After overnight incubation, the reaction solution sample was extracted, and the peptide fragments in the system were qualitatively analyzed using liquid chromatography-tandem mass spectrometry (Waters Xevo G2-XS QTof). The analytical results clearly showed that all the designed enzymes described in this invention can specifically cleave the Aβ42 peptide. The specific mass spectrometry fragmentation data are as follows: PP507-S1 cleaved Aβ42 into fragments with molecular weights of 2068 Da and 2463 Da; PP532-S1 cleaved Aβ42 into fragments with molecular weights of 744 Da, 1736 Da, and 2068 Da; DP622-S2 cleaved Aβ42 into fragments with molecular weights of 400 Da and 4131 Da; DP221-S3 cleaved Aβ42 into fragments with molecular weights of 744 Da, 858 Da, 3674 Da, and 3787 Da; OP609-S2 cleaved Aβ42 into fragments with molecular weights of 400 Da and 4131 Da; OP669-S2 cleaved Aβ42 into fragments with molecular weights of 400 Da and 4131 Da.

[0043] Figures 2 to 7The mass spectrum shows the hydrolysis products of the zinc-dependent metalloproteinase. It can be seen that after incubation with the different recombinant proteases described in this invention, the intact Aβ42 monomer signal is significantly degraded, and selective polypeptide fragments are clearly detected in the mass spectrum. This experimental result directly demonstrates that the zinc-dependent metalloproteinase designed de novo in this invention can recognize and cleave the core pathogenic polypeptide Aβ42 of Alzheimer's disease under physiological conditions.

[0044] Example 4: Cryo-electron microscopy structural analysis of zinc-dependent metalloproteinases PP509-S1, DP622-S2, and DP221-S3 To overcome the molecular weight limitations of de novo metalloproteinases with small molecular weights in high-resolution single-particle cryo-electron microscopy, this embodiment employs a rigid fusion strategy. The metalloproteinase is inactivated and mutated, then ligated to a structural aid tag (i.e., a large molecular weight scaffold protein) for high-contrast characterization. This structural aid tag serves only as an observational tool to enhance spatial size and assist in structural resolution; it does not alter the core catalytic activity, binding pocket structure, or substrate selectivity of the metalloproteinase itself.

[0045] To directly confirm the tight binding of the enzyme active site to the target substrate at atomic resolution, this embodiment prepared a protein complex sample containing the substrate. First, a protein complex solution was prepared containing a fusion of the protease and the scaffold protein (SEQ ID NO: 13-15) concentrated to 5 mg / mL. This fusion was obtained by fusing the protease with the scaffold protein (multi-helical bundle protein) for use in cryo-electron microscopy high-resolution imaging. The fusion protein was prepared by expression using an *E. coli* prokaryotic expression system followed by purification. Subsequently, a 1.5-fold molar excess of Aβ42 peptide and an equimolar concentration of zinc ions were added to the system to ensure the metal catalytic site achieved the correct pre-organized state. Cryo-sample preparation was performed using the FEIVitrobot Mark IV semi-automated sample preparation device. The internal ambient temperature of the Vitrobot instrument was precisely set to 8°C, the relative humidity to 100%, and the ink absorption time to 3 seconds. In the sample preparation chamber, 2.5 μL of the composite solution was dropped onto an amorphous nickel-titanium alloy porous mesh (ANTcryo M02-Cu300-1.2 / 1.3) that had undergone glow discharge treatment. A rapid freezing operation was then performed, and the prepared mesh was transferred to liquid nitrogen for safekeeping before analysis. The rapidly frozen mesh was then mounted on a 300 kV Titan Krios G4 cryo-transmission electron microscope (Thermo Fisher Scientific) equipped with a Falcon4i detector for observation. Image acquisition was performed using EPU automated software (Thermo Fisher Scientific), with a magnification of 215,000X and a defocus range between -1.0 μm and -2.0 μm. The total electron dose per image stack was approximately 50 e. - / Ų, resulting in a pixel size of 0.57 Å for the final high-resolution photomicrograph.

[0046] Cryo-electron microscopy data were processed using cryoSPARC, and the density map of the reconstructed electron microscopy results is shown below. Figure 8 See the resolution result image. Figure 9 As can be seen, the zinc-dependent metalloproteinase designed in this invention forms a highly stable and well-matched complex with the target polypeptide substrate, and the overall resolution reaches 3.13 Å, 2.98 Å, and 3.44 Å. The high-resolution cryo-electron microscopy density maps clearly reveal the atomic-level interaction interface between the enzyme's active site and the target substrate. This atomic-level structure confirms that the de novo designed recombinant protease of this invention can accurately recognize and tightly bind to the target substrate (Aβ42 polypeptide), achieving the expected spatial conformation and sequence selectivity, and demonstrating its potential as an Aβ42 degrader from a physical mechanism perspective.

[0047] Amino acid sequence of OP669-S2 MKIIIEGENPGKEELKKLMEVLQKIFSDKKGEIIVRIYSTPEEGREMAERILERIGEELAKLPGLSPAARQFVLDYVRRLLDATLSVPGAVGGFALANDDWVLIGVLAPSDTPLDDLIFTLFHEATHVALFDMVRRGKIPDGDRFFDKRELDGFVVGVEGINAEAIADVFGALVVAALRDDLSAAQARQLAIDRSRRIAELGERLGLPVHAEGGRKAVELASSLSDEQVEEIRGLLREALASGDREKFEAAIMMLVDALR (SEQ ID NO: 1) Amino acid sequence of PP507-S1 RQQNLDVLDKIVVYPEGEYDREKADAIVDALAWIPLKYLQALLDAGVKIVLANSEELLELLKKYPRLRDLIRALIEVAKMTVGVGDGGDKRAYINIEGSVEGVRLLALHETAHVIDDVVLNSISLSEEFLKMFEELKKMLLEALKKKPEALKNYELNKKILDKLENEELTPEEIRELVRKMKENIVGLSAAYLGTYPEEAFAWGFAAYALRRGGDEEGLERFRRYLMRTSGLDPESVEKVLDMLIKMGEWAAEK (SEQ ID NO: 2) Amino acid sequence of PP532-1 MQEARDILDEIVEFPEGEYDREAAQEVVDALARIPIEYLRALKEAGVKIRLTDLETAKAYAKQAALERGDEETYKRIDKAGIVGVGGELPDGIGAVIGWRPEYARRHPEDVRFLALHETAHVIDDVVLDDISYSERFLEIFKKYGEAIGEMAAKAFPRMGSREEILAYLGTYPDEAFATSMAMYWLDVPEANEDPKVKEAIDQIVALADEALRRRRA (SEQID NO: 3) Amino acid sequence of DP622-S2 RNLELARAADVTVTVADTPEEMYEAAKVAVETVRELAAGPRRDEYVALAERLFRTGIERGGIAGIAIYADGRRRVFVVAPSDASDEALIYALAHELAHLIIAEDLERRGLPLSAVPPGVVEGLADVFGATAYAAYLELKGEKVTLEKWREMQLRLAEETERIGREAGLEAHVEGGRIAAEIARRTNEEEAQKLIEEVKPLVEFILELLRQARES(SEQ ID NO: 4) DP221-S3 amino acid sequence EVTVLTASSPDEIPAIAERVLELTGDREVAERVREAMAELWRALAAMPDVVIGGLALLDYPDVVVLVEGPRPASEEARLLTLAHEIAHGIFARLAREAGVDLSLPEVRAANEGVATFAGLAEMLGVASVEEAARAAFDWYLGDPRARERAARLPVTEEQLRRMLEEVEKLAEIYRRAREQGLLPAEFTDAHLGGTEVATRILRFVGEARGL(SEQ ID NO: 5) OP609-S2 amino acid sequence MEIEIRGEKAKTLRELAETLKKIFSDYEGKILVYIYDTPEEGRRFAEEMVREARDFAREADPSDAERVEEYVRRLMEATLSVPGAIGGFALADEKRVLIAVLAPSDAPPEDLLFAMIHEATHIALYRMYNGELMKDPVRVEAIADVFGALAVGALREDLNPEEIRRLAVERSRAIAELGERLGLPVHARGGRLARELAEGLSDEEIERAVELIRKALKTGDRKNFEEAIRILVEALD(SEQ ID NO: 6) TV-S1 amino acid sequence MGHHHHHHHHSSGLEVLFQGPGGTVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGGSGGGSHHQKLVFFAEDGGGSGGGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 7) Amino acid sequence of TV-S2 (SEQ ID NO: 8) TV-S3 amino acid sequence (SEQ ID NO: 9) TV-S4 amino acid sequence MGHHHHHHHHSSGLEVLFQGPGGTVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGGSGGGSNKGAIIGLMVGGVVIAGGGSGGGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 10) Aβ42 amino acid sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 11).

[0048] Amino acid sequence of PP507-S1 fused to a basal scaffold protein Amino acid sequence of DP622-S2 fusion with basic scaffold protein Amino acid sequence of DP221-S3 fusion with basic scaffold protein

Claims

1. A protease, said protease being: (a) Having an amino acid sequence selected from any one of SEQ ID NO: 1 to 6; or (b) A protein derived from (a) whose amino acid sequence in (a) has been substituted, deleted or added with one or more amino acids and has the activity of degrading amyloid β (Aβ).

2. The protease according to claim 1, wherein: The protease has the amino acid sequence shown in SEQ ID NO: 1, or The protease has the amino acid sequence shown in SEQ ID NO: 2, or The protease has the amino acid sequence shown in SEQ ID NO: 3, or The protease has the amino acid sequence shown in SEQ ID NO: 4, or The protease has the amino acid sequence shown in SEQ ID NO: 5, or The protease has the amino acid sequence shown in SEQ ID NO:

6.

3. A polynucleotide encoding the protease according to claim 1 or 2.

4. A recombinant vector comprising the polynucleotide according to claim 3.

5. The recombinant vector according to claim 4, wherein, The recombinant vector is an expression vector.

6. A recombinant cell comprising the protease according to claim 1 or 2, the polynucleotide according to claim 3, or the recombinant vector according to claim 4.

7. A composition comprising the protease according to claim 1 or 2, the polynucleotide according to claim 3, the recombinant vector according to claim 4, or the recombinant cell according to claim 6.

8. The use of the protease according to claim 1 or 2, the polynucleotide according to claim 3, the recombinant vector according to claim 4, the recombinant cell according to claim 6, or the composition according to claim 7 in the preparation of amyloid β, in particular, an Aβ42 degrading agent or scavenger.

9. The use of the protease according to claim 1 or 2, the polynucleotide according to claim 3, the recombinant vector according to claim 4, the recombinant cell according to claim 6, or the composition according to claim 7 in the preparation of a medicament for the prevention or treatment of diseases caused by abnormal aggregation of amyloid β, in particular Aβ42.

10. The application according to claim 9, wherein, The disease in question is Alzheimer's disease.

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