Synthesis and application of a truncated peptide of type IV collagen

Abstract

The present invention provides a method for screening active truncated peptides of type IV collagen using a bioinformatics method, the truncated peptides obtained by the method and their application. The present invention also provides a method for obtaining the truncated peptides by fermentation using a yeast expression system.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to a type IV collagen truncated peptide and an application thereof. Background Art

[0002] Proteins are core molecules of life activities, and they undertake multiple functions such as catalysis, biological signal transmission, structural support, material transport and immune defense. They participate in and regulate almost all biological processes through their unique three-dimensional structure and functional diversity. For example, enzymes act as catalysts to accelerate biochemical reactions; receptor proteins mediate signal transmission between cells; structural proteins such as collagen and keratin provide structural support for tissues and cells. Collagen is the most abundant protein in mammalian tissues, accounting for 30% of the total protein content of the body. It is also an important component of the extracellular matrix, which can maintain the structure and function of tissues and organs such as skin, cartilage, tendons, ligaments and internal organs. Collagen has the characteristics of biodegradability, low immunogenicity and promoting cell proliferation and differentiation, and is an ideal biomedical and beauty skin care material.

[0003] There are at least 28 different types of collagen in vertebrates. Unlike most collagens, type IV collagen is only found in the basement membrane (BM). The skin basement membrane separates epidermal basal cells from the underlying connected tissues, provides structural support to cells, and maintains the function of the epidermis and dermis through the regulation of epidermal-dermal cytokines. The basement membrane also has selective permeability, regulating the exchange of substances between the epidermis and dermis, such as nutrients or aging waste. The type IV collagen network structure serves as the core structural skeleton of the basement membrane, providing structural support for cells and extracellular matrix. Type IV collagen has multiple binding sites, including binding sites for laminin, bone morphogenetic protein (BMP), heparin, etc. In addition to providing a scaffold for assembly and mechanical stability, type IV collagen is also an important component of the interaction between cells and the basement membrane. This interaction is directly related to a variety of biological processes including cell adhesion, migration, survival, proliferation and differentiation. With the rise of biosynthesis technology, the application of type IV collagen as food, medicine, personal care products and cosmetics is also gradually increasing. Patent CN118406135A describes the biosynthesis and application of a 281 amino acid fragment of the α4 chain of human type IV collagen.

[0004] However, traditional collagen is mainly extracted from terrestrial animal connective tissue and aquatic processing byproducts through hot water extraction, acid-base hydrolysis and enzymatic hydrolysis. However, the separation and purification process of animal-derived collagen is complicated and the monomer separation is difficult. It may also carry viruses and pose a safety hazard, which to some extent limits the application and development of animal-derived collagen.

[0005] In recent years, with the rapid development of computational biology, bioinformatics and artificial intelligence technologies, researchers have begun to focus on de novo protein design technology. De novo design uses algorithms to generate new protein sequences and structures from scratch. This provides great possibilities for designing new proteins or protein fragments according to specific functional requirements.

[0006] However, existing de novo design technologies still have some problems and defects. First, the designed proteins are not stable enough. Many computer-designed proteins cannot fold correctly in the actual environment, resulting in loss of function or instability. This is mainly due to the limitations of existing algorithms in predicting receptor folding and stability, and the inability to fully consider the intrinsic physicochemical properties of the receptor.

[0007] Secondly, the function prediction is inaccurate. When simulating protein-protein interactions, computational models may not fully capture complex biochemical features, resulting in functional predictions that are inconsistent with actual conditions. This inaccuracy limits the practical application of newly designed receptors. Summary of the invention

[0008] The present invention establishes a completely new protein de novo design and screening method, and uses the above method to screen and obtain truncated peptides of type IV collagen with excellent anti-aging activity, and achieves its efficient expression in a yeast expression system.

[0009] In a first aspect, the present invention provides a polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 1 or a variant amino acid sequence obtained by substituting, deleting or inserting one or more amino acids in SEQ ID NO: 1.

[0010] In certain embodiments, the polypeptide does not comprise a full-length type IV collagen sequence.

[0011] In certain embodiments, the polypeptide is a truncated peptide of type IV collagen.

[0012] In certain embodiments, the type IV collagen is native type IV collagen.

[0013] In certain embodiments, the type IV collagen is at least 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to native type IV collagen.

[0014] In certain embodiments, the native type IV collagen has the amino acid sequence shown in SEQ ID NO: 21.

[0015] In certain embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1 and has a length of 31-50 amino acids; preferably, a length of 31-45 amino acids; more preferably, a length of 31-40 amino acids.

[0016] In certain embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1 and has a length of 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, or 40 amino acids.

[0017] In certain embodiments, the deletion comprises a continuous deletion of up to 5, up to 4, up to 3, or up to 2 amino acids at the N-terminus of SEQ ID NO: 1 and / or a continuous deletion of up to 5, up to 4, up to 3, or up to 2 amino acids at the C-terminus of SEQ ID NO: 1.

[0018] In certain embodiments, the deletion includes a total of no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids deleted from the N-terminus and C-terminus of SEQ ID NO: 1.

[0019] In certain embodiments, the variant has an activity comparable to or better than that of SEQ ID NO:1.

[0020] In certain embodiments, the activity comprises mitochondrial protection, skin tissue morphology protection and / or promotion of endogenous anti-aging-related protein expression.

[0021] In certain embodiments, the activity is exhibited under UV radiation.

[0022] In certain embodiments, the anti-aging-associated proteins include type IV collagen, type VII collagen, type XVII collagen and / or laminin 5.

[0023] In certain embodiments, the polypeptide comprises a modification.

[0024] In certain embodiments, the modification includes N-terminal modification, C-terminal modification, side chain modification, amino acid modification, backbone modification, etc.

[0025] The second aspect of the present invention provides a polynucleotide encoding the polypeptide according to the first aspect of the present invention.

[0026] In certain embodiments, the polynucleotide further comprises a sequence encoding a purification tag and / or a signal peptide sequence.

[0027] In certain embodiments, the purification tag comprises a His tag.

[0028] In certain embodiments, the signal peptide is a hybrid signal peptide consisting of an Ost1 secretion signal peptide and a pro signal peptide.

[0029] In certain embodiments, the signal peptide comprises the amino acid sequence shown in SEQ ID NO:2.

[0030] In certain embodiments, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 3 or a degenerate sequence thereof.

[0031] The third aspect of the present invention provides a vector comprising the polynucleotide described in the first aspect of the present invention.

[0032] In certain embodiments, the vector is an integrating plasmid.

[0033] In certain embodiments, the integrating plasmid is a yeast integrating plasmid.

[0034] In certain embodiments, the expression cassette of the integration plasmid comprises the polynucleotide described in the first invention of the present invention.

[0035] In certain embodiments, the expression cassette further comprises a methanol-inducible promoter of the AOX1 gene and / or a transcription terminator of the AOX1 gene.

[0036] In certain embodiments, the backbone sequence of the integration plasmid includes a geneticin (G418) resistance gene cassette, an ampicillin resistance gene cassette, and a Col E1 replicon.

[0037] In certain embodiments, the backbone sequence comprises the nucleotide sequence shown in SEQ ID NO: 9 or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 9;

[0038] In certain embodiments, the vector comprises the nucleotide sequence shown in SEQ ID NO: 18 or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18.

[0039] The fourth aspect of the present invention provides a cell, which comprises the polynucleotide described in the second aspect of the present invention or the vector described in the third aspect of the present invention.

[0040] In certain embodiments, the cell is a eukaryotic cell; more preferably, the eukaryotic cell is a yeast host cell.

[0041] The fifth aspect of the present invention provides a composition comprising the polypeptide described in the first aspect of the present invention.

[0042] In certain embodiments, the composition is formulated into a dosage form suitable for administration by a route selected from oral administration, external application and injection.

[0043] The sixth aspect of the present invention provides the use of the polypeptide described in the first aspect of the present invention or the composition described in the fifth aspect of the present invention in the preparation of cosmetics, health products, food additives or medicines.

[0044] The seventh aspect of the present invention provides a method for producing the polypeptide described in the first aspect of the present invention, comprising fermenting and culturing the cell described in the fourth aspect of the present invention, and isolating and purifying the polypeptide from the culture.

[0045] In certain embodiments, the cell is a yeast cell.

[0046] In certain embodiments, methanol induction is performed during fermentation expression in yeast.

[0047] In certain embodiments, the polypeptide is obtained by centrifugation.

[0048] In certain embodiments, purification of the polypeptide is performed by affinity chromatography.

[0049] The eighth aspect of the present invention provides a method for designing and screening functional protein active peptides, comprising the following steps:

[0050] 1) Identification of functional protein binding receptors;

[0051] 2) Binding site analysis: Use the protein docking model to analyze the potential binding sites on the functional protein that bind to the receptor;

[0052] 3) Peptide design: Use diffusion model to design the backbone according to the binding site;

[0053] 4) Sequence generation: Use the ProteinMPNN model to generate the corresponding peptide amino acid sequence for the peptide backbone designed in step 3);

[0054] 5) Structure prediction: perform three-dimensional structure prediction on the sequence generated in step 4);

[0055] 6) Result screening: Screening is performed based on the structure prediction results of step 5) to obtain the first candidate peptide group;

[0056] 7) Sequence alignment and candidate optimization: Align the first candidate peptide group obtained in step 6) with the known functional protein sequence, evaluate the similarity and difference between the candidate peptide sequence and the known functional protein sequence, sort them according to the similarity, and screen to obtain the target number of second candidate peptide groups with high similarity to the known functional protein sequence;

[0057] 8) Experimental verification: Perform functional verification on the second candidate peptide group obtained in step 7), rank the peptides by performance based on the experimental data, and obtain one or more target active peptides.

[0058] In certain embodiments, in step 1), potential functional protein receptors are identified by searching protein databases and literature retrieval.

[0059] In certain embodiments, the protein docking model in step 2) uses the CB-Dock 2 tool to perform binding site analysis on the identified receptor; preferably, the CB-Dock 2 is based on cavity detection and the AutoDock Vina algorithm, and is capable of identifying potential binding sites on the protein surface.

[0060] In certain embodiments, in step 3), 1000-3000 polypeptide backbones are designed for each binding site.

[0061] In certain embodiments, in step 4), 2-6 polypeptide sequences are generated for each polypeptide backbone. Preferably, 4 polypeptide sequences are generated for each polypeptide backbone.

[0062] In certain embodiments, AlphaFold2 is used in step 5) to predict the three-dimensional structure of the protein.

[0063] In some embodiments, the step 6) is screened according to the prediction score provided by AlphaFold2, and the screening criteria include: PAE_interaction < 5 Å, binder_aligned_rmsd < 1.5 Å, target_aligned_rmsd < 1.5 Å, pae_binder < 5 Å, pae_interaction < 10 Å, pae_target < 5 Å, plddt_binder > 90 and / or plddt_target > 90.

[0064] In certain embodiments, the experiment in step 8) includes a binding ability experiment, a cell experiment and / or an animal experiment.

[0065] In certain embodiments, the functional protein is type IV collagen; the experiments in step 8) include experiments on binding ability with integrin receptors, and cell adhesion and migration experiments.

[0066] In certain embodiments, the active peptides obtained by screening are as described in the first aspect of the present invention.

[0067] The ninth aspect of the present invention provides the application of the method described in the eighth aspect of the present invention in cosmetic raw material development, drug development and / or basic research.

[0068] The present invention has the following advantages over the prior art:

[0069] 1. An efficient and systematic method for designing protein active peptides is provided. By using advanced computational models (such as RFDiffusion, ProteinMPNN, AlphaFold2) and high-throughput experimental platforms (such as Bota Freeway), the whole process from receptor recognition, binding site analysis, peptide design, sequence generation, structure prediction, screening, experimental verification to model optimization is realized. The design method of the present invention has the following characteristics: 1) High efficiency: the automated and parallelized process significantly improves the efficiency of design and verification; 2) High accuracy: multiple screening and optimization ensure the high quality of the final candidate peptides; 3) Scalability: the method can be applied to other protein systems besides type IV collagen, and has broad application prospects. The design method of the present invention accelerates the development of new protein receptors, and also provides strong technical support for the fields of biomedicine and biotechnology, and promotes the development of related scientific research and industry.

[0070] 2. A 31-amino acid fragment on the 5α chain of type IV collagen was successfully screened. The obtained active truncated peptide was functionally verified, proving that the truncated peptide has a strong mitochondrial protective effect, has a good protective effect on UV-irradiated skin tissue morphology, and can effectively protect the effects of UV radiation on endogenous type IV, type VII, type XVII collagen in skin cells and Laminin 5 in skin cells, thereby achieving the effect of resisting photoaging.

[0071] 3. The yeast expression system of active truncated peptides of type IV collagen was realized with high efficiency, overcoming the defects of the existing technology of difficult collagen extraction and potential safety hazards. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The integrated plasmid map of collagen Col4A5 truncated peptide is shown.

[0073] Figure 2 The electrophoresis diagram shows colony PCR verification of the integration of collagen Col4A5 truncated peptide plasmid into the genome.

[0074] Figure 3 The yield of Col4A5 truncated peptides at different fermentation time points was detected by SDS-PAGE.

[0075] Figure 4 The yield of Col4A5 truncated peptide at different fermentation time points was shown in BCA assay.

[0076] Figure 5 Shows mitochondrial membrane potential analysis, BC is the untreated control group, UVA (10J / cm²) is the negative control group, (UVA 10J / cm²+TGF-β1) is the positive control group, and (UVA10J / cm² +100ppm recombinant type IV collagen truncated peptide) is the sample group.

[0077] Figure 6 It showed that 0.01% collagen IV truncated peptide could significantly promote the thickness of the epidermis after UVA+UVB irradiation.

[0078] Figure 7 It showed that 0.01% of type IV collagen truncated peptide sample could significantly increase the density of dermal fibroblasts.

[0079] Figure 8 It showed that 0.01% of type IV collagen truncated peptide sample could significantly promote the expression of type IV collagen.

[0080] Fig. 9 It showed that 0.01% of type IV collagen truncated peptide sample could significantly promote the expression of collagen VII.

[0081] Fig.10 It showed that 0.01% type IV collagen sample could significantly promote the expression of collagen XVII.

[0082] Fig.11 It showed that 0.01% of type IV collagen truncated peptide sample could significantly promote the expression of laminin.

[0083] Figure 6-Figure 11 BC in it is the untreated control group; UVR is the group of combined irradiation of UVA (30J / cm2) and UVB (50mJ / cm2) for 35min; UVR+(VC+VE) is the group after the above irradiation, VC (100 μg / mL) + VE (7 μg / mL) is used continuously for one week; UVR+100ppm Col IV is the sample group after the above irradiation, 100ppm Col IV sample is used continuously for one week. DETAILED DESCRIPTION

[0084] The following definitions are provided to facilitate understanding of the present invention by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. Preferred materials and methods are described herein, but any methods and materials similar to or equivalent to those described herein may be used in the practice of the present invention's testing, but. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0085] As used in this article, "collagen" is the main component of animal connective tissue and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein, and even more than 80% in some organisms. There are many types of collagen, and more than 16 types of collagen have been discovered. According to the distribution and functional characteristics of collagen, it can be divided into interstitial collagen, basement membrane collagen and pericellular collagen. Interstitial collagen includes types I, II and III collagen. Type I collagen is mainly distributed in tissues such as skin, tendons, and bones. It is most abundant in the human body and has the effects of moisturizing, firming, and brightening the skin. Type II collagen is mainly produced by chondrocytes and is also distributed in eye tissues, which can maintain the function of cartilage and eyes; type III collagen is mainly distributed in the dermis, cardiovascular and gastrointestinal tract, and has the function of maintaining tissue elasticity. Basement membrane collagen usually refers to type IV collagen, which is mainly distributed in the basement membrane of the skin and kidney; pericellular collagen usually refers to type V collagen, which is mainly distributed in the amnion and some embryonic tissues.

[0086] As used herein, "type IV collagen" is a fibrous protein that, unlike most collagens, is found only in the basement membrane (BM) and consists of six genes encoding different α chains, named α1(IV) to α6(IV). These six chains specifically interact and assemble to form three different heterotrimers, namely α1α1α2, α3α4α5, and α5α5α6. The α1(IV) and α2(IV) chains are called "classical" chains because they were identified first and are found in the basement membrane of all tissues, while the other four chains are distributed in specific tissues during development. For example, the α3(IV), α4(IV), and α5(IV) chains are found in the glomerular basement membrane (GBM) of the kidney, lung, testis, and eye, while the α5(IV) and α6(IV) chains are found in the basement membrane of the skin, smooth muscle, and kidney.

[0087] As used herein, "polypeptide" refers to a polymer of amino acid residues. A "polypeptide" may be modified (e.g., phosphorylated or glycosylated) or unmodified. A "polypeptide" may include "conservative substitutions", which, in relation to an amino acid sequence, refer to replacement of an amino acid residue by a different amino acid residue containing a side chain having similar physicochemical properties. For example, conservative substitutions may be made between amino acid residues having hydrophobic side chains, between amino acid residues having neutral hydrophilic side chains, between amino acid residues having aromatic side chains, between amino acid residues having acidic side chains, or between amino acid residues having basic side chains. As is known in the art, conservative substitutions generally do not cause significant changes in the conformational structure of the protein, and therefore the biological activity of the protein may be retained.

[0088] As used herein, "truncated peptide", "truncated fragment", "truncated peptide", "truncated fragment" or "truncated body" all refer to a peptide formed by removing one or more amino acids from one or both ends of a wild-type polypeptide. In the present application, "truncated peptide", "truncated fragment", "truncated peptide", "truncated fragment" or "truncated body" does not include the full length of the corresponding wild-type polypeptide, but may have one or more amino acid substitutions, deletions, insertions or modifications compared to the truncated form of the wild-type polypeptide. For example, "type IV collagen truncated peptide" may include a peptide formed by removing one or more amino acids from one or both ends of wild-type type IV collagen, and may also include a peptide having one or more amino acid substitutions, deletions, insertions or modifications compared to the truncated form of wild-type type IV collagen. In the present application, "type IV collagen truncated peptide" is a polypeptide fragment truncated to a length of less than 500, less than 400, less than 300, less than 200, less than 100, less than 50, or less than 40 amino acids relative to the wild-type collagen Col4A5 (UniProt ID P29400). For example, the truncated peptide comprises amino acids 509-539 of Col4A5.

[0089] As used herein, the term "substitution" refers to the replacement of at least one amino acid in an amino acid sequence by a different amino acid. The term "insertion" refers to the insertion of at least one additional amino acid into an amino acid sequence. The "insertion" usually includes 1 or 2 amino acids, and may also include about 3 to 5 or even more amino acids. The above "substituted" amino acids may be conservative or non-conservative. The above "substituted" or "inserted" amino acids may be natural or non-natural. The term "deletion" refers to the removal of at least one amino acid from an amino acid sequence, and the removal may occur at both ends or in the middle of the amino acid sequence, and the deletion may be continuous or non-continuous.

[0090] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparison and comparison sequences. "Percentage identity", "percentage homology", "sequence identity" or "sequence homology" etc. means the percentage of identical residues between the amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule compared. For example, a sequence A that is "at least 85% identical" to sequence B means that sequence A comprises at least 85%, such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical residues to sequence B. For these calculations, preferably by specific mathematical models or computer programs (i.e., "algorithms"), addressing comparisons are performed in the case of allowing spaces. When calculating percentage identity, the sequences compared are typically compared in a manner that gives the maximum match between the sequences.

[0091] As used herein, "vector" refers to a DNA molecule that is responsible for transferring DNA fragments (target genes) to recipient cells in genetic engineering recombinant DNA technology. Vectors can be divided into cloning vectors and expression vectors. Cloning vectors are mainly used to clone and amplify DNA fragments. There are mainly plasmid vectors, phage vectors, phagemid vectors, and viral vectors. In addition to the basic elements of cloning vectors, expression vectors also have control elements necessary for transcription and translation, such as promoters and terminators.

[0092] As used herein, the term "control element" refers to a nucleic acid sequence necessary for expression of a polynucleotide encoding a truncated peptide of the invention. The control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) to the polynucleotide encoding the polypeptide, or native or exogenous to each other. Such control elements include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control elements include a promoter, and transcription and translation termination signals.

[0093] As used herein, "integrative plasmid" refers to a DNA sequence containing a selectable marker and at least one target gene expression cassette. Before being transformed into yeast, the integrative plasmid is linearized, which allows its sequence to be inserted into the yeast genomic region. Yeast integrative plasmids are genetic units that cannot replicate autonomously outside the chromosome and must be integrated into the yeast chromosome to replicate.

[0094] As used herein, "yeast host cell", "yeast cell" means any yeast host cell that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or a recombinant expression vector comprising a polynucleotide of the present invention. The yeast host cell can be a Pichia, Candida, Hansenula, Kluyveromyces, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

[0095] As used herein, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0096] As used herein, the term "protein docking model" means: When a potential receptor is identified, CB Dock 2 is used for binding site analysis. CB Dock 2 combines depression detection and AutoDock Vina algorithms to predict possible binding sites by identifying depressions on the surface of the target protein. This method first uses geometric shape analysis and molecular surface features to identify potential binding sites, and then evaluates the binding affinity of each binding site through molecular docking simulation.

[0097] As used in this article, the term "diffusion pool model (RFDiffusion)" is an advanced generative model that uses a diffusion process to generate samples in a high-dimensional space. The original structure is processed by gradually adding noise, and a denoising network obtained through training is used to generate new peptides that meet specific functional requirements. This process not only involves the de novo generation of functionally defined peptide fragments, but also combines the optimization of biocompatibility and physicochemical properties to ensure that the generated peptides have biological activity.

[0098] Design and screening methods of functional protein active peptides

[0099] The present invention provides a method for designing and screening functional protein active peptides, comprising the following steps:

[0100] 1) Identification of functional protein binding receptors;

[0101] 2) Binding site analysis: Use the protein docking model to analyze the potential binding sites on the functional protein that bind to the receptor;

[0102] 3) Peptide design: Use diffusion model to design the backbone according to the binding site;

[0103] 4) Sequence generation: Use the ProteinMPNN model to generate the corresponding peptide amino acid sequence for the peptide backbone designed in step 3);

[0104] 5) Structure prediction: perform three-dimensional structure prediction on the sequence generated in step 4);

[0105] 6) Result screening: Screening is performed based on the structure prediction results of step 5) to obtain the first candidate peptide group;

[0106] 7) Sequence alignment and candidate optimization: Align the first candidate peptide group obtained in step 6) with the known functional protein sequence, evaluate the similarity and difference between the candidate peptide sequence and the known functional protein sequence, sort them according to the similarity, and screen to obtain the target number of second candidate peptide groups with high similarity to the known functional protein sequence;

[0107] 8) Experimental verification: Perform functional verification on the second candidate peptide group obtained in step 7), rank the peptides by performance based on the experimental data, and obtain one or more target active peptides.

[0108] In certain embodiments, in step 1), potential functional protein receptors are identified by searching a protein database (such as Uniprot) and retrieval of literature; the potential functional protein receptors are those that have been reported, predicted, or predictable in prior art literature or protein databases.

[0109] In certain embodiments, the protein docking model in step 2) uses the CB-Dock 2 tool to perform binding site analysis on the identified receptor; preferably, the CB-Dock 2 tool identifies potential binding sites on the protein surface based on cavity detection and the AutoDock Vina algorithm.

[0110] In certain embodiments, the potential binding sites identified by the protein docking model in step 2) are key sites for protein-protein interactions.

[0111] In certain embodiments, in step 3), 1000-3000 polypeptide backbones are designed for each binding site using RFDiffusion (diffusion model); for example, about 1000, about 2000, or about 3000 polypeptide backbones are designed for each binding site.

[0112] In certain embodiments, in step 3), a total of about 2000-3000 frameworks are designed for all binding sites.

[0113] In certain embodiments, in step 4), the ProteinMPNN model is used to generate 2-6 polypeptide sequences for each polypeptide backbone; for example, 2, 3, 4, 5, or 6 polypeptide sequences are generated for each polypeptide backbone.

[0114] In certain embodiments, AlphaFold2 is used in step 5) to predict the three-dimensional structure of the protein.

[0115] In certain embodiments, the screening in step 6) is performed based on the prediction score provided by AlphaFold2.

[0116] In certain embodiments, the screening criteria include: PAE_interaction < 5 Å, binder_aligned_rmsd < 1.5 Å, target_aligned_rmsd < 1.5 Å, pae_binder < 5 Å, pae_interaction <10 Å, pae_target < 5 Å, plddt_binder > 90 and / or plddt_target > 90.

[0117] In certain embodiments, the target number in step 7) is 50-200, such as about 100.

[0118] In certain embodiments, the experiment in step 8) includes a binding ability experiment, a cell experiment and / or an animal experiment.

[0119] In certain embodiments, the method for designing and screening functional protein active peptides may optionally further include step 9) data analysis and feedback and step 10) iterative design.

[0120] In certain embodiments, the data analysis and feedback includes in-depth analysis of the experimental results to identify successful and failed modes. For successful modes: analyze the sequence and structural features of the polypeptides with excellent performance to find commonalities; for failed modes: analyze the possible reasons for the polypeptides that did not achieve the expected effect, such as sequence instability, structural prediction deviation, etc. Feedback the analysis results to the model to adjust the model parameters.

[0121] In certain embodiments, ProteinMPNN is optimized based on the characteristics of successful polypeptides, the sequence generation strategy is optimized, and the model weights are adjusted.

[0122] In certain embodiments, AlphaFold2 is optimized based on the analysis results to improve the accuracy of structure prediction.

[0123] In certain embodiments, the iterative design includes conducting a new round of polypeptide design, screening and verification based on the optimized model.

[0124] The design and screening method of functional protein active peptides provided by the present invention can be widely used in the de novo design of various functional proteins, including but not limited to collagen, elastin, fibronectin, etc.

[0125] For example, the elastin peptide of Seq ID NO.1 in the applicant's prior patent CN118290568A is obtained by using the design and screening method of the functional protein active peptide of the present invention. The elastin peptide has the ability to significantly promote the synthesis of its own elastin gene and fibronectin gene, and at the same time significantly promote the synthesis of collagen IV and VII genes in the dermal-epidermal junction layer; anti-UV experiments have shown that the recombinant human elastin fragment can protect elastin, collagen I, IV and VII from being destroyed by ultraviolet rays and other excellent properties.

[0126] For another example, the four truncated fragments of human fibronectin named 0417, 0418, 0419 and 0420 in the applicant's prior patent CN117298257B were also obtained using the design and screening method of functional protein active peptides of the present invention. The above-mentioned fibronectin fragments have the effects of improving the skin barrier and increasing cell adhesion.

[0127] Design and screening methods of type IV collagen truncated peptides

[0128] The present invention provides a method for screening active truncated peptides of type IV collagen, comprising the following steps: 1) identification of type IV collagen binding receptors; 2) binding site analysis: using a protein docking model to analyze potential binding sites on type IV collagen with integrin receptors; 3) polypeptide design: using a diffusion model to design a skeleton according to the binding site; 4) sequence generation: using a ProteinMPNN model to generate a corresponding polypeptide amino acid sequence for the polypeptide skeleton designed in step 3); 5) structure prediction: performing three-dimensional structure prediction on the sequence generated in step 4); 6) result screening: screening to obtain a first candidate polypeptide group according to the structure prediction result of step 5); 7) sequence alignment and candidate optimization: comparing the first candidate polypeptide group obtained in step 6) with known type IV collagen, evaluating the similarity and difference between the candidate polypeptide sequence and the known type IV collagen, sorting by similarity, and screening to obtain a target number of second candidate polypeptide groups with high similarity to the known functional protein sequence; 8) experimental verification: performing functional verification on the second candidate polypeptide group obtained in step 7), and ranking the polypeptides by performance according to experimental data to obtain one or more active type IV collagen truncated peptides.

[0129] The screening method for active truncated peptides of type IV collagen provided by the present invention is:

[0130] 1) Receptor identification: Identify potential collagen IV receptors through protein database searches and extensive literature searches.

[0131] 2) Binding site analysis: The CB-Dock 2 tool was used to perform binding site analysis on the identified integrin receptors. Based on cavity detection and the AutoDock Vina algorithm, CB-Dock 2 can identify potential binding sites on the surface of proteins. Twelve hotspots were selected from the two pockets of collagen IV, which are considered to be the key sites where collagen IV and integrin may interact.

[0132] 3) Peptide design: RFDiffusion (diffusion model) was used to design peptide fragments around the 12 identified hotspots. RFDiffusion's design_ppi (protein-protein interaction) method was used to generate a total of 2,000 peptide backbones for the hotspots.

[0133] 4) The ProteinMPNN (Protein Message Passing Neural Network) model was used to generate the corresponding amino acid sequences for the 2,000 peptide backbones designed and generated. The parameter --seqs_per_struct 4 was used when running ProteinMPNN to generate 4 different amino acid sequences for each peptide backbone. A total of 8,000 candidate sequences (2,000 backbones × 4 sequences) were generated.

[0134] 5) AlphaFold2 was used to predict the three-dimensional structure of the generated 8,000 amino acid sequences.

[0135] 6) Result screening:

[0136] Among the obtained structural models, screening was performed based on the prediction scores provided by AlphaFold2, with particular attention paid to the PAE values ​​of the protein interaction regions in the Predicted Alignment Error (PAE) matrix. The screening conditions are as follows:

[0137] •PAE_interaction < 5 Å: Select models with PAE values ​​less than 5, indicating high confidence in the predictions in the interaction region.

[0138] •binder_aligned_rmsd < 1.5 Å: The root mean square deviation (RMSD) of the binder, which indicates the spatial difference between the designed binder and the target structure. The smaller the value, the more similar the structures are.

[0139] •target_aligned_rmsd < 1.5 Å: The root mean square deviation of the target and designed combination after alignment, reflecting the structural similarity between the two.

[0140] •pae_binder < 5 Å: The predicted alignment error (PAE) of the binder, which is used to evaluate the accuracy of the relative position information between atoms within the binder.

[0141] •pae_interaction < 10 Å: PAE values ​​for protein-protein interaction regions to ensure high-quality interaction predictions.

[0142] •pae_target < 5 Å: PAE value of the target protein, which reflects the prediction error of the overall structure and ensures the stability of the target structure.

[0143] •plddt_binder > 90: The local deep learning score of the combination (Predicted Local DistanceDifference Test), which reflects the overall reliability of the combination. The higher the value, the higher the credibility.

[0144] •plddt_target > 90: Local deep learning score of the target protein, evaluating the reliability of the target structure.

[0145] •plddt_total > 70: The total local deep learning score of the combination of body and target, reflecting the reliability of the overall prediction.

[0146] Using the above screening criteria, 1030 high-quality structural models were selected from 8000 candidate sequences.

[0147] 7) Sequence alignment and candidate optimization

[0148] Sequence alignment tools such as BLAST (Basic Local Alignment Search Tool) and Clustal Omega were used to evaluate the similarity and difference between candidate sequences and known sequences. 100 candidate sequences with high similarity to known functional sequences were screened out for subsequent experimental verification.

[0149] 8) High-throughput experimental verification: Use high-throughput experimental platforms (such as Bota Freeway) to perform functional verification on the selected sequences, determine the binding kinetic parameters of the peptides and integrin receptors, and evaluate the effectiveness of the peptides in biological functions through cell experiments (such as cell adhesion and migration experiments). Finally, a 31-amino acid fragment (GFPGQKGEKGQAGATGPKGLPGIPGAPGAPG) that specifically binds to the 5α chain of type IV collagen was successfully identified.

[0150] Type IV collagen truncated peptide

[0151] The present invention provides a type IV collagen truncated peptide comprising the amino acid sequence shown in SEQ ID NO: 1 or a variant amino acid sequence obtained by substituting, deleting or inserting one or more amino acids based on SEQ ID NO: 1.

[0152] GFPGQKGEKGQAGATGPKGLPGIPGAPGAPG (SEQ ID NO: 1).

[0153] In certain embodiments, the type IV collagen truncated peptide comprises the amino acid sequence shown in SEQ ID NO:1.

[0154] In certain embodiments, the type IV collagen truncated peptide consists of the amino acid sequence shown in SEQ ID NO:1.

[0155] In certain embodiments, the type IV collagen truncated peptide comprises the amino acid sequence shown in SEQ ID NO: 1 and has a length of 31-50 amino acids. Preferably, the length is 31-45 amino acids; more preferably, the length is 31-40 amino acids; more preferably, the length is 31, 32, 33, 34, 35, 36, 37, 38, 39 amino acids.

[0156] In certain embodiments, the type IV collagen truncated peptide has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with native type IV collagen (e.g., UniProtID P29400), wherein the native type IV collagen has an amino acid sequence as shown in SEQ ID NO: 21.

[0157] In certain embodiments, the deletion occurs at both ends or in the middle of SEQ ID NO:1.

[0158] In certain embodiments, the deletion is continuous or discontinuous.

[0159] In certain embodiments, the deletion comprises a continuous deletion of up to 5, up to 4, up to 3, or up to 2 amino acids at the N-terminus of SEQ ID NO: 1 and / or a continuous deletion of up to 5, up to 4, up to 3, or up to 2 amino acids at the C-terminus of SEQ ID NO: 1.

[0160] In certain embodiments, the sum of amino acids deleted from the N-terminus and the C-terminus of SEQ ID NO: 1 does not exceed 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0161] In certain embodiments, the type IV collagen truncated peptide may be modified or unmodified.

[0162] In certain embodiments, the modification includes N-terminal modification, C-terminal modification, side chain modification, amino acid modification, backbone modification, etc.

[0163] In certain embodiments, the modification includes cyclization, glycosylation, phosphorylation, N-methylation, myristoylation and palmitoylation, polyethylene glycol (PEG) modification, etc.

[0164] In certain embodiments, the cyclization modification is divided into side chain-side chain type, terminal-side chain type, and terminal-terminal type.

[0165] In certain embodiments, the cyclization modification forms collagen microspheres.

[0166] In certain embodiments, the variant of type IV collagen truncated peptide has an activity comparable to or superior to that of SEQ ID NO:1.

[0167] Method for constructing recombinant bacteria

[0168] The present invention provides a method for constructing a yeast cell expressing a type IV collagen truncated peptide, comprising introducing a recombinant expression vector into a yeast cell, wherein the recombinant expression vector comprises a polynucleotide encoding the collagen truncated peptide herein. The recombinant expression vector may comprise a nucleotide sequence as shown in SEQ ID NO: 18. The yeast may be Pichia pastoris, preferably Pichia pastoris BG11 strain.

[0169] The construction of the recombinant bacteria of the present invention includes the steps of: 1) designing the synthetic gene of collagen Col4A5 truncated peptide, 2) constructing the expression cassette, 3) constructing the integration plasmid, and 4) transforming yeast.

[0170] The collagen Col4A5 truncated peptide synthetic gene of the present invention encodes a truncated peptide shown in SEQ ID NO: 1 with a signal peptide placed at the N-terminus and a purification tag connected to the C-terminus.

[0171] In certain embodiments, the signal peptide is a hybrid signal peptide composed of the Ost1 secretion signal peptide of Saccharomyces cerevisiae and the pro signal peptide in the pre-pro signal peptide of mating factor α; preferably, the hybrid signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2.

[0172] In certain embodiments, the purification tag is selected from HIS-Tag, GST-Tag, MBP-Tag, and NusA-Tag.

[0173] In certain embodiments, the synthetic gene is optimized according to a codon optimization technique. Preferably, the codon optimization technique comprises the "Codon Optimization Tool" from Integrated DNA Technologies, Inc.

[0174] The collagen Col4A5 truncated peptide expression box of the present invention comprises a synthetic gene, a promoter and a terminator.

[0175] In certain embodiments, the promoter is a methanol-inducible promoter; preferably, the methanol-inducible promoter is derived from the AOX1 gene promoter, and its sequence is shown in SEQ ID NO:4.

[0176] In certain embodiments, the terminator is derived from the AOX1 gene transcription terminator, and its sequence is shown in SEQ ID NO:5.

[0177] The collagen Col4A5 truncated peptide expression cassette of the present invention is integrated into the yeast genome via an integration plasmid, wherein the integration plasmid comprises an expression cassette and a plasmid skeleton.

[0178] In certain embodiments, the backbone of the integration plasmid includes: a geneticin (G418) resistance gene cassette (SEQ ID NO: 6), a Col E1 replicon (SEQ ID NO: 7), and an ampicillin resistance gene cassette (SEQ ID NO: 8).

[0179] In certain embodiments, the backbone of the integration plasmid comprises the nucleotide sequence shown in SEQ ID NO:9.

[0180] In certain embodiments, the integration plasmid is Figure 1 As shown, it comprises the nucleotide sequence shown in SEQ ID NO:18.

[0181] The integration plasmid of the present invention is linearized and then transformed into a yeast strain and integrated into the yeast genome.

[0182] In certain embodiments, the integration plasmid is linearized using the PmeI restriction enzyme, which cuts in the middle of the methanol-inducible promoter sequence of the AOX1 gene.

[0183] Recombinant bacteria fermentation method

[0184] The present invention provides a method for producing type IV collagen truncated peptides by fermenting yeast transformed with an integration plasmid.

[0185] In certain embodiments, the fermentation culture is preceded by a seed culture.

[0186] In certain embodiments, the fermentation medium comprises the basal medium of Table 1 and the additives of Table 2.

[0187] In certain embodiments, seeds are added to the fermentation medium, the initial OD of the fermentation culture is 0.2-0.4, and after 20-26 hours of fermentation culture, glycerol is added, and the initial addition amount is 23-33 ml / kg / h. The amount of glycerol added is subsequently controlled according to the dissolved oxygen to maintain the dissolved oxygen at about 20%.

[0188] In certain embodiments, the seeds are added to the fermentation medium with an initial OD of 0.3. After fermentation for 22-24 hours, glycerol is added at an initial amount of 28 ml / kg / h. The amount of glycerol added is subsequently controlled according to the dissolved oxygen to maintain the dissolved oxygen at about 20%.

[0189] In certain embodiments, the cells are cultured to a wet weight of 330-380 g / L and then switched to methanol for induction. The initial amount of methanol added is 0.4-0.6 ml / kg / h. The rate of methanol addition is subsequently adjusted according to the dissolved oxygen to maintain the dissolved oxygen at about 40%. The induction time is 150-190 h.

[0190] In certain embodiments, the cells are cultured to a wet weight of 330-380 g / L and then switched to methanol for induction. The initial amount of methanol added is 0.5 ml / kg / h. The rate of methanol addition is subsequently adjusted according to the dissolved oxygen to maintain the dissolved oxygen at about 40%. The induction time is 170 h.

[0191] The invention provides a separation and purification method after fermentation culture.

[0192] In certain embodiments, the separation includes taking the cultured bacterial solution, centrifuging the bacterial solution in a low-temperature centrifuge, and retaining the supernatant.

[0193] In certain embodiments, the separating comprises filtering the supernatant through a 0.45 μm filter membrane.

[0194] In certain embodiments, the purification method comprises affinity column chromatography.

[0195] In certain embodiments, the chromatography includes: flowing the sample through a Ni column at a rate of 1 ml / min; balancing the column with Buffer A (20mM PB, pH7.5); eluting with 20mM, 300mM, or 500mM imidazole; and analyzing the eluted sample by SDS-PAGE gel electrophoresis to determine whether the target protein is present.

[0196] Example

[0197] The present invention will be more readily understood with reference to the following examples, which are included only to illustrate certain aspects and embodiments of the present invention and are not intended to limit the present invention.

[0198] Unless otherwise stated, the reagents used in this example are all commercially available materials or conventional materials.

[0199] Example 1 Screening of Type IV Collagen Truncated Peptides

[0200] The potential receptors of type IV collagen were identified by using bioinformatics methods, potential binding peptide sites were identified by using a protein docking model, peptide fragments were designed by using a diffusion pool model, and then compared with the amino acid sequences of 6 different chains of type IV collagen to screen out potential peptides, and the target peptides were screened by wet experiments. Specifically, the method includes the following steps:

[0201] 1) Receptor identification: Identify potential type IV collagen receptors by searching protein databases (such as Uniprot) and extensive literature searches.

[0202] 2) Binding site analysis: The CB-Dock 2 tool was used to perform binding site analysis on the identified integrin receptors. Based on cavity detection and the AutoDock Vina algorithm, CB-Dock 2 can identify potential binding sites on the surface of proteins. Specifically, 12 hotspots were selected from the two pockets of type IV collagen. These hotspots are considered to be the key sites where type IV collagen and integrins may interact. Through in-depth analysis of these hotspots, important structural information is provided for subsequent peptide design.

[0203] 3) Peptide design:

[0204] Around the 12 identified hotspots, RFDiffusion (diffusion model) was used to perform a new design of peptide fragments. RFDiffusion is a generative model based on a diffusion process that can generate peptide structures that meet specific functional requirements in high-dimensional space. Specifically, the design_ppi (protein-protein interaction) method of RFDiffusion was used to generate a total of 2,000 peptide backbones for the 12 hotspots.

[0205] 4) Sequence generation:

[0206] The ProteinMPNN (Protein Message Passing Neural Network) model was used to generate the corresponding amino acid sequences for the generated peptide backbones. The parameter --seqs_per_struct4 was used when running ProteinMPNN, which generated 4 different amino acid sequences for each peptide backbone. In this way, a total of 8,000 candidate sequences were generated (2,000 backbones × 4 sequences).

[0207] Features and workflow of ProteinMPNN:

[0208] •Graph neural network: represents the polypeptide skeleton as a graph structure, where nodes represent amino acids and edges represent interactions.

[0209] •Sequence optimization: The model predicts the most suitable amino acid sequence based on the backbone structure and optimizes the sequence to improve stability and functionality.

[0210] •Parallel generation: Generate multiple sequences at one time, increasing the diversity of candidate sequences.

[0211] 5) Structure prediction: AlphaFold2 was used to predict the three-dimensional structure of the generated 8,000 amino acid sequences. AlphaFold2 is a protein structure prediction model based on deep learning, which can accurately predict the three-dimensional folding structure of proteins. The initial guess mode of AlphaFold2 is used in the present invention to improve the prediction efficiency.

[0212] How AlphaFold2 works:

[0213] •Multiple sequence alignment (MSA): Use evolutionary information to construct a multiple sequence alignment of the target sequence.

[0214] •Neural network prediction: Through deep neural network, predict structural information such as distance map and angle distribution.

[0215] •Structure reconstruction: Reconstruct the three-dimensional structure of the protein based on the predicted structural information.

[0216] 6) Result screening:

[0217] Among the obtained structural models, screening was performed based on the prediction scores provided by AlphaFold2, with particular attention paid to the PAE values ​​of the protein interaction regions in the Predicted Alignment Error (PAE) matrix. The screening conditions are as follows:

[0218] •PAE_interaction < 5 Å: Select models with PAE values ​​less than 5, indicating high confidence in the predictions in the interaction region.

[0219] •binder_aligned_rmsd < 1.5 Å: The root mean square deviation (RMSD) of the binder, which indicates the spatial difference between the designed binder and the target structure. The smaller the value, the more similar the structures are.

[0220] •target_aligned_rmsd < 1.5 Å: The root mean square deviation of the target and designed combination after alignment, reflecting the structural similarity between the two.

[0221] •pae_binder < 5 Å: The predicted alignment error (PAE) of the binder, which is used to evaluate the accuracy of the relative position information between atoms within the binder.

[0222] •pae_interaction < 10 Å: PAE values ​​for protein-protein interaction regions to ensure high-quality interaction predictions.

[0223] •pae_target < 5 Å: PAE value of the target protein, which reflects the prediction error of the overall structure and ensures the stability of the target structure.

[0224] •plddt_binder > 90: The local deep learning score of the combination (Predicted Local DistanceDifference Test), which reflects the overall reliability of the combination. The higher the value, the higher the credibility.

[0225] •plddt_target > 90: Local deep learning score of the target protein, evaluating the reliability of the target structure.

[0226] •plddt_total > 70: The total local deep learning score of the combination of body and target, reflecting the reliability of the overall prediction.

[0227] Using the above screening criteria, 1030 high-quality structural models were selected from 8000 candidate sequences.

[0228] 7) Sequence alignment and candidate optimization:

[0229] The 1030 candidate sequences screened were compared with known type IV collagen in the literature. Sequence alignment tools such as BLAST (Basic Local Alignment Search Tool) and Clustal Omega were used to evaluate the similarities and differences between the candidate sequences and the known sequences.

[0230] Comparison process:

[0231] •BLAST alignment: Align the candidate sequence with known sequences in the database to obtain a similarity score.

[0232] •Multiple sequence alignment: Use Clustal Omega to align multiple sequences and identify conserved regions and variable sites.

[0233] •Screening criteria: Select candidate sequences with high similarity to known functional sequences.

[0234] Through comparison analysis, 100 candidate sequences with high similarity to known functional sequences were screened out for subsequent experimental verification.

[0235] 8) Experimental verification:

[0236] The company's high-throughput experimental platform, Bota Freeway, was used to express and functionally verify the 100 candidate peptides screened.

[0237] Experimental process:

[0238] • Peptide synthesis: synthesize 100 candidate peptides to ensure that the purity and quality meet the experimental requirements.

[0239] •Binding experiment: Surface plasmon resonance (SPR) technology is used to determine the binding kinetic parameters (such as binding constant and dissociation constant) between the peptide and integrin receptor.

[0240] •Functional evaluation: Evaluate the effectiveness of the peptide in biological function through cell experiments (such as cell adhesion and migration experiments).

[0241] •Data collection: Record the experimental data of each peptide, including binding affinity, functional activity, etc.

[0242] Result analysis:

[0243] •Success rate: Among the 100 candidate peptides, a portion was found to exhibit the expected high affinity and biological activity.

[0244] • Performance ranking: Based on the experimental data, the peptides were ranked by performance, and the best performing fragment of 31 amino acids on the 5α chain of type IV collagen (SEQ ID NO: 1, GFPGQKGEKGQAGATGPKGLPGIPGAPGAPG) was selected for further study.

[0245] Example 2 Synthesis and purification of truncated peptides of the original protein Col4A5

[0246] 2.1 Construction of the integrated plasmid expression system for collagen Col4A5 truncated peptide

[0247] Collagen Col4A5 truncated peptide synthesis gene

[0248] The collagen Col4A5 truncated peptide (SEQ ID NO: 1, GFPGQKGEKGQAGATGPKGLPGI PGAPGAPG) is a fragment (amino acids 509-539) of human collagen Col4A5 (UniProt ID P29400), which was screened by the method described in Example 1. To facilitate purification, a His tag (-GSSHHHHHH) was attached to the C-terminus. The peptide was designed to be expressed in a secretory manner by the yeast Komagataella phaffii (formerly Pichia pastoris). A hybrid signal peptide (SEQ ID NO: 2) consisting of the Ost1 secretion signal peptide of Saccharomyces cerevisiae and the pro signal peptide in the pre-pro signal peptide of mating factor α was placed upstream of the collagen Col4A5 truncated peptide sequence to direct secretion. In order to improve the expression efficiency, the product gene sequence was designed and optimized using codon optimization technology; the specific method is as follows: the nucleotide sequence encoding the signal peptide and the collagen Col4A5 truncated peptide with a His tag at the C-terminus was codon optimized using the "Codon Optimization Tool" on Integrated DNA Technologies, Inc. The codon-optimized gene sequence (SEQ ID NO: 3) was then synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.

[0249] Collagen Col4A5 truncated peptide expression cassette

[0250] The collagen Col4A5 truncated peptide expression cassette includes the methanol-inducible promoter of the AOX1 gene (SEQ ID NO: 4, Komagataella phaffii CBS 7435 chromosome IV nucleotides 238,036-238,974, GenBank LT962479.2), the synthetic gene sequence encoding the collagen peptide as described above (SEQ ID NO: 3), and the transcription terminator of the AOX1 gene (SEQ ID NO: 5, Komagataella phaffii CBS 7435 chromosome IV nucleotides 240,967-241,316, GenBank LT962479.2). The integration plasmid is used to integrate the collagen Col4A5 truncated peptide expression cassette into the yeast genome.

[0251] Construction of integration plasmid

[0252] The backbone of the integration plasmid includes: a geneticin (G418) resistance gene cassette (SEQ ID NO: 6), a Col E1 replicon (SEQ ID NO: 7), and an ampicillin resistance gene cassette (SEQ ID NO: 8). The backbone sequence was synthesized by Beijing Liuhe BGI Technology Co., Ltd. (SEQ ID NO: 9).

[0253] To construct the integration plasmid, the backbone sequence (SEQ ID NO: 9) was amplified using primers backbone-F and backbone-R (SEQ ID NO: 10 and SEQ ID NO: 11).

[0254] The genomic DNA of the BG11 strain (PS 10011) of BioGrammatics Inc. was prepared using the TIANamp Yeast DNA Extraction Kit (DP307-02) of TIANGEN BIOTECH (BEIJING) Co, LTD. Using this genomic DNA as a template, the methanol-inducible promoter sequence (SEQ ID NO: 4) of the AOX1 gene was amplified using primers Pro-F and Pro-R (SEQ ID NO: 12 and SEQ ID NO: 13).

[0255] The synthetic collagen Col4A5 truncated peptide gene (SEQ ID NO: 3) was amplified using primers Col4-F and Col4-R (SEQ ID NO: 14 and SEQ ID NO: 15). The Col4A5 truncated peptide gene template was synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd. (SEQ ID NO: 9) (SEQ ID NO: 3).

[0256] Using genomic DNA of the BG11 strain (PS 10011) from BioGrammatics Inc. as a template, the transcription terminator (SEQ ID NO: 5) of the AOX1 gene was amplified using primers Ter-F and Ter-R (SEQ ID NO: 16 and SEQ ID NO: 17).

[0257] The amplified products were ligated using NEB's NEBuilder HiFi DNA Assembly Premix Kit and transformed into E. coli DH5α (DL1003M from WEIDI Bio, Inc.). Transformed E. coli clones were screened on Luria-Bertani (LB) medium containing 100 mg / L ampicillin sodium. The assembled sequence of the plasmid was verified by Sanger sequencing. The plasmid was named pCol4A5pep (SEQ ID NO: 18, Figure 1 ).

[0258] Construction of expression strain of collagen Col4A5 truncated peptide

[0259] The integration plasmid pCol4A5pep was purified from E. coli culture using the TIANprep Mini Plasmid Kit (DP103-02) from Tianjin Biotechnology (Beijing) Co., Ltd. The plasmid was linearized using the PmeI restriction enzyme (ThermoFisher ER1341), which cuts in the middle of the methanol-inducible promoter sequence of the AOX1 gene.

[0260] 100 ng of linearized plasmid was used to transform the BG11 strain (PS 10011, BioGrammatics Inc.) by electroporation. Preparation of competent cells and electroporation followed the protocol in "Protein Expression in Pichia pastoris" (Methods in Enzymology, 2021 Vol 660, 53-80).

[0261] Transformants were selected on YPD solid medium containing 1 g / L G418.

[0262] The integration of the integrated plasmid into the AOX1 gene in the transformant genome was verified by colony PCR using primer 5U (SEQ ID NO: 19), which binds to the genomic sequence upstream of the AOX1 gene inducible promoter, and primer 3C (SEQ ID NO: 20), which binds to the plasmid-specific sequence of the collagen Col4A5 truncated peptide expression vector. The integrated transformant should produce a 1.9 kb PCR product ( Figure 2 ). The PCR product was sequenced by Sanger sequencing to confirm the presence of the collagen expression vector. The steps of colony PCR are from "Protein Expression in Pichia pastoris", Enzymology Methods, Volume 660, 2021, Pages 53-80. The verified transformant strain was named Strain-Col4A5pep.

[0263] Collagen Col4A5 truncated peptide yeast fermentation process

[0264] The composition of the fermentation medium is shown in Tables 1 and 2. The medium was sterilized at 121°C for 30 minutes, cooled to 28°C for use, and PTM1 and HMP were added after cooling.

[0265] Table 1 Fermentation medium composition

[0266]

[0267] Table 2 PTM1 formulation

[0268]

[0269] The monoclonal strain was added to 50 mL YPD medium and cultured overnight at 28°C and 250 rpm. The next day, the first-level shake flask seeds were transferred to 100 mL YPD medium, OD = 0.5, and cultured at 28°C and 250 rpm for 24 hours as seeds for the tank. The seeds for the tank were added to the above fermentation medium, with an initial OD = 0.3. After 22-24 hours of fermentation culture, glycerol was added. The initial addition amount was 28 ml / kg / h. The amount of glycerol added was subsequently controlled according to the dissolved oxygen to maintain the dissolved oxygen at about 20%. The cells were cultured to a wet weight of 330-380 g / L, and then switched to methanol for induction. The initial addition amount of methanol was 0.5 ml / kg / h. The methanol addition rate was subsequently adjusted according to the dissolved oxygen to maintain the dissolved oxygen at about 40%. The induction time was 170 hours. The yield of Col4A5 at different fermentation time points was detected by SDS-PAGE and BCA. The results are as follows: Figure 3 and Figure 4 shown.

[0270] Purification of collagen Col4A5 truncated peptide

[0271] Take the cultured bacterial solution, centrifuge it in a low-temperature centrifuge, and retain the supernatant.

[0272] The supernatant obtained above was filtered with a 0.45 μm filter membrane and then passed through a 5 ml affinity chromatography column for protein purification. The steps are as follows:

[0273] 1) The sample was passed through the Ni column at a rate of 1 ml / min;

[0274] 2) Equilibrate the column with Buffer A (20 mM PB, pH 7.5);

[0275] 3) Elution with 20mM, 300mM, or 500mM imidazole.

[0276] 4) Perform SDS-PAGE analysis on the eluted samples to determine whether the target protein is present.

[0277] Example 3 Protective effect of collagen Col4A5 truncated peptide on mitochondria

[0278] The protective effect of Col4A5 truncated peptide on mitochondria was verified by mitochondrial membrane potential (ΔΨM) analysis.

[0279] Experimental groups:

[0280] Table 3 Analysis of mitochondrial membrane potential (ΔΨM) groups

[0281]

[0282] Experimental Procedure

[0283] Primary human dermal fibroblasts (HDFs) (lifeline, #FC-0024) were expanded in fibroblast medium at 37°C, 95% air humidity and 5% CO2. When the cells grew to 80-90% confluence, they were trypsinized and plated into 96-well plates. When the cells reached 90% confluence in the 96-well plates, they were irradiated with UVA light at an intensity of 10J / cm². After UVA irradiation, HDFs were immediately treated according to the experimental group and incubated for a further 24 hours. The cell culture medium was removed and 2 μM (final concentration) of JC-1 dye was added. The cells were incubated for 20 minutes at 37°C and 5% CO2. The dye solution was then removed and the cells were washed twice with buffer. The JC-1 fluorescence of the test cultures (including the control) was immediately analyzed. For the quantification method, red fluorescence (excitation 568 nm / emission 590 nm) and green fluorescence (excitation 485 nm / emission 535 nm) were measured using a multiplate reader. Then, the ratio of red fluorescence to green fluorescence was determined. At the same time, fluorescence images were captured for live cells on coverslips.

[0284] Note: In apoptotic and dead cells, the dye will appear green as a monomer; in live, non-apoptotic cells, the dye aggregates in the mitochondria and appears red. At higher ΔΨM, JC-1 dye monomers aggregate to form red fluorescent "J-aggregates" that accumulate in the mitochondria. J-aggregates form a broad excitation spectrum and have a maximum emission peak at about 590nm. When the JC-1 dye is at low mitochondrial concentration or low ΔΨM, it exists as a monomer with an emission wavelength of 535nm.

[0285] result

[0286] The results are as follows Figure 5 The results showed that compared with the blank control group (BC), the mitochondrial membrane potential of the negative control group (10J / cm² UVA) decreased significantly ( ### p<0.001), indicating that the model of mitochondrial damage in fibroblasts caused by UVA radiation was successfully established; compared with the negative control group, the mitochondrial membrane potential of fibroblasts treated with 100ppm recombinant type IV collagen truncated peptide was significantly improved ( *** p<0.001), and the effect was comparable to that of the positive control TGF-β1, indicating that recombinant type IV collagen has a strong mitochondrial protective effect.

[0287] Example 4: Effect of collagen Col4A5 truncated peptide on the expression of aging-related proteins

[0288] The promoting effect of Col4A5 truncated peptide on the expression of aging-related proteins was analyzed through in vitro skin experiments.

[0289] Experimental Grouping :

[0290] Table 4 Grouping of ex vivo skin experiments

[0291]

[0292] Experimental Procedure :

[0293] 1) Tissue processing

[0294] Freshly obtained skin tissue (Guangdong Boxi Biotechnology Co., Ltd.) was immersed in 75% alcohol and washed for 30 seconds, and then washed three times with sterile PBS buffer; after that, the skin was cut into 24±2mm 2 Place the tissue block with the epidermis facing up and the dermis facing down into a culture mold, then transfer the culture mold into a 6-well plate, add 3.7 mL of culture medium to each well, and culture in a 37°C, 5% CO2 incubator, changing the medium every day.

[0295] 2) Drug administration

[0296] After one day of in vitro skin tissue culture, the drugs and irradiation treatments were carried out according to the experimental groups and corresponding treatment conditions. UVA (irradiation dose was UVA (30 J / cm 2 ) and UVB (50mJ / cm 2 ) irradiation (irradiation time is about 35 minutes), and after irradiation, the test substance is smeared on the tissue surface (before smearing the test substance on the second day, the test substance remaining on the surface needs to be removed), and fresh culture medium is replaced at the same time. After 4 consecutive days, only medication is performed, that is, the test substance is smeared on the tissue surface every day (before smearing the test substance on the second day, the test substance remaining on the surface needs to be removed), and this continues for 3 days.

[0297] 3) Tissue morphology test

[0298] Skin tissues for tissue morphology detection were obtained and fixed with 4% paraformaldehyde. After fixation for 24 hours, H&E staining was performed, and photos were taken under a microscope for observation, collection and analysis.

[0299] 4) Immunofluorescence detection

[0300] After the administration, the skin tissue was fixed with 4% paraformaldehyde, and then embedded and sliced. The paraffin sections were placed in a 70℃ baking machine and baked for 4 hours. The sections were soaked in xylene for 10 minutes, replaced with xylene and then soaked for 10 minutes, soaked in anhydrous ethanol for 5 minutes, soaked in 95% ethanol for 5 minutes, and soaked in 75% ethanol for 5 minutes. Washed with PBS buffer 3 times, 5 minutes each time. The paraffin sections were placed in 0.01M sodium citrate antigen repair solution, repaired with high pressure, and the sections were removed after cooling. Washed with PBS buffer solution 3 times, 5 minutes / time. Add 1 drop of 3% H2O2 to each section and incubate at room temperature for 30 minutes to block the activity of endogenous peroxidase. Washed with PBS buffer solution 3 times, 5 minutes / time. Add serum homologous to the secondary antibody and block at 37℃ for 60 minutes without rinsing. Add the primary antibody working solution (as follows: Anti-collagen IV antibody, Catalog No. ab6311; Anti-Collagen VII antibody, Catalog No. ab6312; Anti-Collagen XVII antibody, Catalog No. ab186415; Anti-Laminin 5 antibody, Catalog No. ab78286; all from abcam) and incubate at 4°C overnight. Wash with PBS buffer 3 times, 5 min / time. Add the secondary antibody working solution (goat anti-rabbit IgG, Catalog No. ab150077; goat anti-mouse IgG, Catalog No. ab150117; all from abcam) and incubate at room temperature for 1 hour. Wash with PBS buffer 3 times, 5 min / time. After the secondary antibody incubation, wash with PBS buffer 3 times, 5 min / time. Shake off the PBS solution attached to the slide, add 100 μL of Hochest working solution to each slice, and incubate at room temperature for 5 min. Wash with PBS buffer 3 times, 5 min / time. Wipe off the PBS solution with absorbent paper, seal the slide with a drop of antifade agent, and take photos with a fluorescence microscope (20×) within 24 hours.

[0301] Experimental Results

[0302] 1) Tissue morphology experimental results

[0303] The results of epidermal living cell layer thickness are as follows Figure 6 Compared with the blank control group (BC), the average thickness of the epidermal living cell layer in the negative control group (UVR irradiation) decreased significantly ( ## p<0.01), indicating that the stimulation conditions of UVR radiation were effective; compared with the negative control group, the thickness of the epidermal living cell layer treated with 100ppm recombinant type IV collagen truncated peptide was significantly increased ( **p<0.01), the improvement rate was 63.34%, which was equivalent to the positive control effect, indicating that recombinant type IV collagen has a good protective effect on the skin tissue morphology irradiated by UV radiation.

[0304] 2) Dermis density results

[0305] like Figure 7 As shown, compared with the blank control group (BC), the density of dermal fibroblasts in the negative control group (UVR irradiation) was significantly decreased ( ## p<0.01), indicating that the stimulation conditions of UVR radiation were effective; compared with the negative control group, the density of dermal fibroblasts treated with 100ppm recombinant type IV collagen truncated peptide was significantly increased ( ** p<0.01), the improvement rate was 140.82%, which was equivalent to the positive control effect, indicating that recombinant type IV collagen has a good protective effect on the skin tissue morphology irradiated by UV radiation.

[0306] 3) Fluorescence staining experimental results

[0307] The results of type IV collagen fluorescence staining were as follows Figure 8 Compared with the blank control group (BC), the type IV collagen content in the negative control group (UVR irradiation) was significantly decreased ( ## p<0.01), indicating that the stimulation conditions of UVR radiation were effective; compared with the negative control group, 100ppm recombinant type IV collagen truncated peptide significantly increased the endogenous type IV collagen content in skin cells ( ** p<0.01), the improvement rate was 72.09%, indicating that recombinant type IV collagen can effectively protect the effect of UV radiation on endogenous type IV collagen in skin cells, thereby achieving the effect of resisting photoaging.

[0308] The results of type VII collagen fluorescence staining are as follows Fig. 9 Compared with the blank control group (BC), the type VII collagen content in the negative control group (UVR irradiation) was significantly decreased ( ## p<0.01), indicating that the stimulation conditions of UVR radiation were effective; compared with the negative control group, 100ppm recombinant type IV collagen truncated peptide significantly increased the content of endogenous type VII collagen in skin cells ( ** p<0.01), the improvement rate was 80.49%, which was equivalent to the positive control effect, indicating that recombinant type IV collagen can effectively protect the effect of UV radiation on endogenous type VII collagen in skin cells, thereby achieving the effect of resisting photoaging.

[0309] The results of type XVII collagen fluorescence staining are as follows Fig.10Compared with the blank control group (BC), the type XVII collagen content in the negative control group (UVR irradiation) was significantly decreased ( ## p<0.01), indicating that the stimulation conditions of UVR radiation were effective; compared with the negative control group, 100ppm recombinant type IV collagen significantly increased the content of endogenous type XVII collagen in skin cells ( ** p<0.01), the improvement rate was 66.67%, which was equivalent to the positive control effect, indicating that recombinant type IV collagen can effectively protect the effect of UV radiation on endogenous type XVII collagen in skin cells, thereby achieving the effect of resisting photoaging.

[0310] The results of laminin 5 fluorescence staining are as follows Fig.11 Compared with the blank control group (BC), the Laminin 5 content in the negative control group (UVR irradiation) decreased significantly ( ## p<0.01), indicating that the stimulation conditions of UVR radiation were effective; compared with the negative control group, 100ppm recombinant type IV collagen truncated peptide significantly increased the content of Laminin 5 in skin cells ( ** p<0.01), the improvement rate was 280%, which was equivalent to the positive control group, indicating that recombinant type IV collagen can effectively protect the effects of UV radiation on Laminin 5 in skin cells, thereby achieving the effect of resisting photoaging.

Claims

1. A polypeptide, characterized in that It consists of the amino acid sequence shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, which has the following activities; the activities include mitochondrial protection, skin tissue morphology protection and / or promotion of endogenous anti-aging related protein expression.

3. The polypeptide according to claim 1 or 2, wherein the anti-aging-related proteins include type IV collagen, type VII collagen, type XVII collagen and / or laminin 5. A polynucleotide encoding the polypeptide according to any one of claims 1 to 3. The polynucleotide according to claim 4 , further comprising a sequence encoding a purification tag and / or a sequence encoding a signal peptide. 6 . The polynucleotide according to claim 5 , wherein the signal peptide is a hybrid signal peptide consisting of an Ost1 secretion signal peptide and a pro signal peptide; and the signal peptide comprises an amino acid sequence shown in SEQ ID NO:

2.

7. The polynucleotide according to claim 6, comprising the nucleotide sequence shown in SEQ ID NO: 3 or a degenerate sequence thereof. A vector comprising the polynucleotide according to any one of claims 4 to 7. The vector according to claim 8 , which is an integrating plasmid.

10. A cell comprising the polynucleotide of any one of claims 4 to 7 or the vector of any one of claims 8 to 9.

11. A composition, characterized in that Comprising the polypeptide according to any one of claims 1 to 3.

12. The composition according to claim 11, which is formulated into a dosage form suitable for administration by a mode selected from oral administration, external application and injection.

13. Use of the polypeptide according to any one of claims 1 to 3 or the composition according to any one of claims 11 to 12 in the preparation of cosmetics, health products or food additives.

14. A method for producing the polypeptide according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: fermenting and culturing the cell according to claim 10, and isolating and purifying the polypeptide from the culture.

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