Synthesis of truncated peptide of type iv collagen and application thereof

Abstract

Provided are a method for screening a truncated bioactive peptide of type IV collagen by using a bioinformatics method, a truncated peptide obtained by means of the method, and an application thereof. Further provided is a method for obtaining the truncated peptide by means of fermentation using a yeast expression system.

Description

[Amended according to Rule 26, December 16, 2024] Synthesis and application of a type IV collagen cleavage peptide. [Amended according to Rule 26, December 16, 2024] Technical Field

[0001] [Amended according to Rule 26 16.12.2024] This disclosure relates to the field of biotechnology, specifically to a type IV collagen truncating peptide and its applications. [Amended according to Rule 26, December 16, 2024] Background Technology

[0002] [Amended according to Rule 26, December 16, 2024] Proteins are the core molecules of life activities, undertaking multiple functions such as catalysis, biological signal transduction, structural support, material transport, and immune defense. Through their unique three-dimensional structure and functional diversity, they participate in and regulate almost all biological processes. For example, enzymes act as catalysts, accelerating biochemical reactions; receptor proteins mediate intercellular signal transduction; 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 body's total protein content. It is also an important component of the extracellular matrix, maintaining the structure and function of tissues and organs such as skin, cartilage, tendons, ligaments, and internal organs. Collagen possesses biodegradability, low immunogenicity, and the ability to promote cell proliferation and differentiation, making it an ideal biomedical and cosmetic skincare material.

[0003] [Amended according to Rule 26, December 16, 2024] Vertebrates contain at least 28 different types of collagen. Unlike most collagens, type IV collagen is found only in the basement membrane (BM). The skin basement membrane separates the basal cells of the epidermis from the underlying connective tissue, providing structural support to the cells and maintaining epidermal-dermal intercellular function through regulation by epidermal-dermal cytokines. The basement membrane also has selective permeability, regulating the exchange of substances between the epidermis and dermis, such as nutrients or waste products. The type IV collagen network structure serves as the core structural framework of the basement membrane, providing structural support for cells and the extracellular matrix. Type IV collagen has multiple binding sites, including binding sites with 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 cell-basement membrane interaction. 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 in food, pharmaceuticals, personal care products, and cosmetics is gradually increasing. Patent CN118406135A describes the biosynthesis and application of 281 amino acid fragments of the α4 chain of human type IV collagen.

[0004] [Amended according to Rule 26, December 16, 2024] However, traditional collagen is mainly extracted from terrestrial animal connective tissues and aquatic processing byproducts through methods such as hot water extraction, acid-base hydrolysis, and enzymatic hydrolysis. Natural collagen obtained from animal tissues and skin extracts accounts for a relatively large proportion. However, the separation and purification process of animal-derived collagen is complex, and monomer separation is difficult. Furthermore, it may carry viruses, posing safety risks and limiting the application and development of animal-derived collagen to some extent.

[0005] [Amended according to Rule 26, December 16, 2024] 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 techniques. De novo design uses algorithms to generate entirely new protein sequences and structures from scratch. This provides enormous possibilities for designing new proteins or protein fragments according to specific functional requirements.

[0006] [Amended according to Rule 26, December 16, 2024] However, existing de novo design techniques still have some problems and shortcomings. First, the designed proteins are not stable enough; many computer-designed proteins fail to fold correctly in real-world environments, leading to loss of function or instability. This is mainly due to the limitations of existing algorithms in predicting receptor folding and stability, and their inability to fully consider the intrinsic physicochemical properties of the receptor.

[0007] [Amended according to Rule 26, December 16, 2024] Secondly, functional predictions are inaccurate. Computational models may fail to adequately capture complex biochemical features when simulating protein-protein interactions, leading to functional predictions that do not match reality. This inaccuracy limits the practical application of newly designed receptors. [Amended according to Rule 26, December 16, 2024] Summary of the Invention

[0008] [Amended according to Rule 26, 16.12.2024] This invention establishes a novel de novo protein design and screening method. Using the above method, a truncated peptide of type IV collagen with excellent anti-aging activity was obtained and its efficient expression in a yeast expression system was achieved.

[0009] [Amended according to Rule 26, 16.12.2024] The first aspect of the present invention provides a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or a variant amino acid sequence obtained by substitution, deletion or insertion of one or more amino acids based on SEQ ID NO: 1.

[0010] [Amended according to Rule 26 16.12.2024] In some embodiments, the polypeptide does not contain a full-length type IV collagen sequence.

[0011] [Amended according to Rule 26 16.12.2024] In some embodiments, the polypeptide is a truncated peptide of type IV collagen.

[0012] [Amended according to Rule 26, 16.12.2024] In some embodiments, the type IV collagen is natural type IV collagen.

[0013] [Amended according to Rule 26, 16.12.2024] In some embodiments, the type IV collagen has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with natural type IV collagen.

[0014] [Amended according to Rule 26 16.12.2024] In some embodiments, the natural type IV collagen has the amino acid sequence shown in SEQ ID NO:21.

[0015] [Amended according to Rule 26, 16.12.2024] In some embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1 and has a length of 31-50 amino acids; preferably, it has a length of 31-45 amino acids; more preferably, it has a length of 31-40 amino acids.

[0016] [Amended according to Rule 26, 16.12.2024] In some embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1 and has a length of 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0017] [Amended according to Rule 26, 16.12.2024] In some embodiments, the deletion includes the sequential deletion of up to 5, up to 4, up to 3, up to 2 amino acids at the N-terminus of SEQ ID NO: 1 and / or the sequential deletion of up to 5, up to 4, up to 3, up to 2 amino acids at the C-terminus of SEQ ID NO: 1.

[0018] [Amended according to Rule 26, 16.12.2024] In some embodiments, the deletion includes the total number of amino acids deleted at the N-terminus and C-terminus of SEQ ID NO: 1 not exceeding 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0019] [Modified according to Rule 26 16.12.2024] In some embodiments, the variant has activity equivalent to or better than SEQ ID NO: 1.

[0020] [Amended according to Rule 26, 16.12.2024] In some embodiments, the activity includes mitochondrial protection, skin tissue morphology protection, and / or promotion of the expression of endogenous anti-aging-related proteins.

[0021] [Amended according to Rule 26 16.12.2024] In some embodiments, the activity is exhibited under UV radiation.

[0022] [Amended according to Rule 26, 16.12.2024] In some embodiments, the anti-aging-related proteins include type IV collagen, type VII collagen, type XVII collagen and / or laminin 5.

[0023] [Amendment to Rule 26, 16.12.2024] In some embodiments, the polypeptide contains modifications.

[0024] [Amended according to Rule 26, 16.12.2024] In some embodiments, the modification includes N-terminal modification, C-terminal modification, side chain modification, amino acid modification, backbone modification, etc.

[0025] [Amended according to Rule 26 16.12.2024] The second aspect of the present invention provides a polynucleotide encoding the polypeptide described in the first aspect of the present invention.

[0026] [Amended according to Rule 26 16.12.2024] In some embodiments, the polynucleotide further comprises a sequence encoding a purification tag and / or a signal peptide sequence.

[0027] [Amended according to Rule 26, 16.12.2024] In some embodiments, the purification tag includes a His tag.

[0028] [Amended according to Rule 26, 16.12.2024] In some embodiments, the signal peptide is a hybrid signal peptide composed of Ost1 secretory signal peptide and pro signal peptide.

[0029] [Amended according to Rule 26 16.12.2024] In some embodiments, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2.

[0030] [Amended according to Rule 26 16.12.2024] In some embodiments, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 3 or a degenerate sequence thereof.

[0031] [Amended according to Rule 26 16.12.2024] A third aspect of the present invention provides a carrier comprising the polynucleotide described in the first aspect of the present invention.

[0032] [Amended according to Rule 26, 16.12.2024] In some embodiments, the vector is an integrative plasmid.

[0033] [Amended according to Rule 26, 16.12.2024] In some embodiments, the integrative plasmid is a yeast integrative plasmid.

[0034] [Amended according to Rule 26 16.12.2024] In some embodiments, the expression cassette of the integrative plasmid includes the polynucleotide described in the first invention.

[0035] [Amended according to Rule 26, 16.12.2024] In some embodiments, the expression cassette further includes a methanol-inducible promoter of the AOX1 gene and / or a transcription terminator of the AOX1 gene.

[0036] [Amended according to Rule 26, 16.12.2024] In some embodiments, the backbone sequence of the integrative plasmid includes a genetic mycotoxin (G418) resistance gene cassette, an ampicillin resistance gene cassette, and a Col E1 replicon.

[0037] [Amended according to Rule 26, 16.12.2024] In some 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%, or 99% identity with SEQ ID NO: 9;

[0038] [Amended according to Rule 26, 16.12.2024] In some 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] [Amended according to Rule 26 16.12.2024] The fourth aspect of the present invention provides a cell comprising the polynucleotide described in the second aspect of the present invention or the carrier described in the third aspect of the present invention.

[0040] [Amended according to Rule 26, 16.12.2024] In some embodiments, the cell is a eukaryotic cell; more preferably, the eukaryotic cell is a yeast host cell.

[0041] [Amended according to Rule 26 16.12.2024] A fifth aspect of the present invention provides a composition comprising the polypeptide described in the first aspect of the present invention.

[0042] [Amended according to Rule 26 16.12.2024] In some embodiments, the composition is formulated as a dosage form suitable for administration by means selected from oral, topical and injection methods.

[0043] [Amended according to Rule 26, 16.12.2024] 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 pharmaceuticals.

[0044] [Amended according to Rule 26, 16.12.2024] 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 cells described in the fourth aspect of the present invention, and isolating and purifying the polypeptide from the culture.

[0045] [Amended according to Rule 26, 16.12.2024] In some embodiments, the cell is a yeast cell.

[0046] [Amended according to Rule 26 16.12.2024] In some embodiments, methanol induction is performed during yeast fermentation expression.

[0047] [Amended according to Rule 26, 16.12.2024] In some embodiments, the polypeptide is obtained by centrifugation.

[0048] [Amended according to Rule 26, 16.12.2024] In some embodiments, the polypeptide is purified by affinity chromatography.

[0049] [Amended according to Rule 26, 16.12.2024] The eighth aspect of this invention provides a method for designing and screening functional protein active peptides, comprising the following steps:

[0050] [Amended according to Rule 26, 16.12.2024] 1) Recognition of functional protein binding receptors;

[0051] [Revised according to Rule 26, 16.12.2024] 2) Binding site analysis: Analyze potential binding sites on functional proteins that bind to receptors using protein docking models;

[0052] [Revised according to Rule 26, 16.12.2024] 3) Peptide design: Scaffold design based on binding sites using a diffusion model;

[0053] [Revised according to Rule 26, 16.12.2024] 4) Sequence generation: Use the ProteinMPNN model to generate the corresponding polypeptide amino acid sequence for the polypeptide backbone designed in step 3);

[0054] [Revised according to Rule 26, 16.12.2024] 5) Structure prediction: Perform three-dimensional structure prediction on the sequence generated in step 4);

[0055] [Revised according to Rule 26, 16.12.2024] 6) Result screening: Based on the structural prediction results of step 5), the first candidate polypeptide group is obtained through screening;

[0056] [Revised according to Rule 26, 16.12.2024] 7) Sequence alignment and candidate optimization: The first candidate polypeptide group obtained in step 6) is aligned with the known functional protein sequence to evaluate the similarity and difference between the candidate polypeptide sequence and the known functional protein sequence. According to the similarity, the target number of second candidate polypeptide groups with high similarity to the known functional protein sequence are selected.

[0057] [Revised according to Rule 26, 16.12.2024] 8) Experimental verification: Perform functional verification on the second candidate polypeptide group obtained in step 7). Based on the experimental data, rank the polypeptides according to their performance and obtain one or more target active polypeptides.

[0058] [Amended according to Rule 26, 16.12.2024] In some embodiments, in step 1), potential functional protein receptors are identified by searching protein databases and literature.

[0059] [Amended according to Rule 26, 16.12.2024] In some embodiments, the protein docking model in step 2) uses the CB-Dock2 tool to analyze the binding sites of the identified receptors; preferably, the CB-Dock2 is based on cavity detection and the AutoDock Vina algorithm and is able to identify potential binding sites on the protein surface.

[0060] [Amended according to Rule 26, 16.12.2024] In some embodiments, step 3) involves designing 1000-3000 polypeptide backbones for each binding site.

[0061] [Amended according to Rule 26, 16.12.2024] In some embodiments, step 4) generates 2-6 polypeptide sequences for each polypeptide backbone. Preferably, 4 polypeptide sequences are generated for each polypeptide backbone.

[0062] [Amended according to Rule 26, 16.12.2024] In some embodiments, step 5) uses AlphaFold2 to predict the three-dimensional structure of the protein.

[0063] [Amended according to Rule 26, 16.12.2024] In some embodiments, step 6) involves screening based on the prediction scores provided by AlphaFold2, with the screening criteria including: plddt_binder>90 and / or plddt_target>90.

[0064] [Amended according to Rule 26, 16.12.2024] In some embodiments, the experiment in step 8) includes binding capacity experiments, cell experiments and / or animal experiments.

[0065] [Amended according to Rule 26, 16.12.2024] In some embodiments, the functional protein is type IV collagen; the experiments in step 8) include experiments on binding ability to integrin receptors, cell adhesion and migration experiments.

[0066] [Amended according to Rule 26, 16.12.2024] In some embodiments, the active peptides obtained by screening are as described in the first aspect of the present invention.

[0067] [Amended according to Rule 26, 16.12.2024] The ninth aspect of the present invention provides the application of the method described in the eighth aspect of the present invention in the development of cosmetic raw materials, drug development and / or basic research.

[0068] [Amended according to Rule 26, 16.12.2024] The present invention has the following advantages over the prior art:

[0069] [Amended according to Rule 26, December 16, 2024] 1. This invention provides an efficient and systematic method for designing bioactive protein peptides. Utilizing advanced computational models (such as RFDiffusion, ProteinMPNN, and AlphaFold2) and high-throughput experimental platforms (such as Bota Freeway), it achieves a complete workflow from receptor identification, binding site analysis, peptide design, sequence generation, structure prediction, screening, experimental validation to model optimization. The design method of this invention has the following advantages: 1) High efficiency: The automated and parallelized workflow significantly improves the efficiency of design and validation; 2) High accuracy: Multiple screenings and optimizations ensure the high quality of the final candidate peptides; 3) Scalability: The method can be applied to other protein systems besides type IV collagen, showing broad application prospects. This invention accelerates the development of novel protein receptors, provides strong technical support for the fields of biomedicine and biotechnology, and promotes the development of related scientific research and industry.

[0070] [Revised according to Rule 26, December 16, 2024] 2. A 31-amino acid fragment of the 5α chain of type IV collagen was successfully screened. The obtained active truncated peptide was functionally verified, demonstrating that the truncated peptide has strong mitochondrial protective effects, good protective effects on skin tissue morphology against UV radiation, and can effectively protect against the effects of UV radiation on endogenous type IV, VII, and XVII collagen in skin cells and skin cell laminin 5, thereby achieving the effect of resisting photoaging.

[0071] [Revised according to Rule 26, 16.12.2024] 3. A yeast expression system for the efficient production of type IV collagen active truncated peptides has been realized, overcoming the shortcomings of existing technologies such as difficulty in collagen extraction and potential safety hazards. [Revised according to Rule 26, December 16, 2024] Attached Figure Description

[0072] [Revised according to Rule 26, 16.12.2024] Figure 1 shows the integrative plasmid map of collagen Col4A5 truncated peptide.

[0073] [Revised according to Rule 26, 16.12.2024] Figure 2 shows the electrophoresis diagram of the integration plasmid of collagen Col4A5 truncated peptide into the genome by colony PCR verification.

[0074] [Revised according to Rule 26, 16.12.2024] Figure 3 shows the yield of Col4A5 truncated peptide at different fermentation time points as detected by SDS-PAGE.

[0075] [Revised according to Rule 26, 16.12.2024] Figure 4 shows the yield of Col4A5 truncated peptide at different fermentation time points as detected by BCA.

[0076] [Revised according to Rule 26, December 2024] Figure 5 shows the mitochondrial membrane potential analysis. BC is the untreated control group, UVA (10 J / cm²). 2 () served as the negative control group, (UVA 10J / cm) 2 +TGF-β1) served as the positive control group, (UVA 10J / cm) 2 The sample group consisted of 100 ppm recombinant type IV collagen truncated peptides.

[0077] [Revised according to Rule 26, 16.12.2024] Figure 6 shows that 0.01% collagen IV cleavage peptide can significantly promote the thickness of the epidermis after UVA+UVB irradiation.

[0078] [Amended according to Rule 26 16.12.2024] Figure 7 shows that 0.01% of the type IV collagen truncated peptide sample can significantly increase the density of dermal fibroblasts.

[0079] [Revised according to Rule 26, 16.12.2024] Figure 8 shows that 0.01% of the type IV collagen truncated peptide sample can significantly promote the expression of type IV collagen.

[0080] [Revised according to Rule 26, 16.12.2024] Figure 9 shows that 0.01% of the type IV collagen truncated peptide sample can significantly promote the expression of collagen VII.

[0081] [Revised according to Rule 26, 16.12.2024] Figure 10 shows that 0.01% of type IV collagen samples can significantly promote the expression of collagen XVII.

[0082] [Amended according to Rule 26, 16.12.2024] Figure 11 shows that 0.01% of the type IV collagen truncated peptide sample can significantly promote laminin expression.

[0083] [Revised according to Rule 26, 16.12.2024] In Figures 6-11, BC is the untreated control group; UVR is the group irradiated with a combination of UVA (30J / cm2) and UVB (50mJ / cm2) for 35min; UVR+(VC+VE) is the group after the above irradiation, followed by continuous use of VC (100μg / mL)+VE (7μg / mL) for one week; UVR+100ppm Col IV is the sample group after the above irradiation, followed by continuous use of 100ppm Col IV for one week. [Revised according to Rule 26, December 2024] Detailed Implementation Method

[0084] [Amended according to Rule 26, 16.12.2024] The following definitions are provided to enable those skilled in the art to understand the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. 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 testing the invention. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0085] [Amended according to Rule 26, December 16, 2024] As used herein, "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 total protein, and even more than 80% in some organisms. There are many types of collagen, with more than 16 types currently discovered. Based on their distribution and functional characteristics, collagen can be divided into interstitial collagen, basement membrane collagen, and peripheral collagen. Interstitial collagen includes type I, II, and III collagen. Type I collagen is mainly distributed in tissues such as skin, tendons, and bones, and is the most abundant in the human body, playing a role in moisturizing, firming, and brightening the skin. Type II collagen is mainly produced by chondrocytes and is also distributed in eye tissues, maintaining the function of cartilage and the eyes; Type III collagen is mainly distributed in the dermis, cardiovascular system, and gastrointestinal tract, maintaining tissue elasticity. Basement membrane collagen usually refers to type IV collagen, which is mainly distributed in the basement membrane of the skin and kidneys; peripheral collagen usually refers to type V collagen, which is mainly distributed in the amnion and some embryonic tissues.

[0086] [Amended according to Rule 26, December 16, 2024] As used herein, “Type IV collagen” is a fibrous protein that, unlike most collagens, is found only in the basement membrane (BM). It consists of six genes encoding distinct α chains, designated α1(IV) through α6(IV). These six chains interact and assemble specifically to form three distinct heterotrimers: α1α1α2, α3α4α5, and α5α5α6. The α1(IV) and α2(IV) chains, being the first to be identified, are referred to as the “classical” chains and are present in the basement membrane of all tissues. The other four chains are distributed in specific tissues during development. For example, the α3(IV), α4(IV), and α5(IV) chains are present in the glomerular basement membrane (GBM) of the kidneys, lungs, testes, and eyes, while the α5(IV) and α6(IV) chains are present in the basement membrane of the skin, smooth muscle, and kidneys.

[0087] [Amended according to Rule 26, 16.12.2024] As used herein, a “polypeptide” refers to a polymer of amino acid residues. A “polypeptide” may be modified (e.g., phosphorylated or glycosylated) or unmodified. A “polypeptide” may contain “conservative substitutions,” which in relation to the amino acid sequence mean that an amino acid residue is replaced by a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions may occur 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 preserve the protein’s biological activity.

[0088] [Amended according to Rule 26, December 16, 2024] As used herein, “truncated peptide,” “truncated fragment,” “shortened peptide,” “shortened fragment,” or “shortened 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 this application, “truncated peptide,” “truncated fragment,” “shortened peptide,” “shortened fragment,” or “shortened body” does not include the full length of its 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, or a peptide with one or more amino acid substitutions, deletions, insertions, or modifications compared to the truncated form of wild-type type IV collagen. In this application, the "Type IV collagen truncated peptide" is a polypeptide fragment truncated from wild-type collagen Col4A5 (UniProt ID P29400) 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. For example, the truncated peptide contains amino acids 509-539 of Col4A5.

[0089] [Amended according to Rule 26, December 16, 2024] As used herein, the term “substitution” means that at least one amino acid in an amino acid sequence is replaced by a different amino acid. The term “insertion” means the insertion of at least one additional amino acid into an amino acid sequence. An “insertion” typically includes one or two amino acids, but may also include about three to five or even more amino acids. The amino acid “substituted” may be conserved or non-conserved. The amino acid “substituted” or “inserted” may be native or non-native. The term “deletion” means the removal of at least one amino acid from an amino acid sequence, which may occur at both ends or in the middle of the amino acid sequence, and the deletion may be continuous or discontinuous.

[0090] [Amended according to Rule 26, 16.12.2024] 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 alignment and comparison of sequences. “Percentage identity,” “percentage homology,” “sequence identity,” or “sequence homology” means the percentage of identical residues among amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared. For example, a sequence A that is “at least 85% identical” to sequence B means that sequence A contains at least 85%, such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of the same residues as sequence B. For these calculations, alignments are preferably addressed using a specific mathematical model or computer program (i.e., an “algorithm”) that allows for gaps. When calculating percentage identity, the compared sequences are typically aligned in a manner that yields the largest possible match between sequences.

[0091] [Amended according to Rule 26, December 16, 2024] As used herein, "vector" refers to a DNA molecule used in recombinant DNA technology to transfer a DNA fragment (target gene) to a recipient cell. Vectors can be divided into cloning vectors and expression vectors. Cloning vectors are mainly used to clone and amplify DNA fragments. They mainly include plasmid vectors, phage vectors, phage particle vectors, and viral vectors. Expression vectors, in addition to the basic elements of cloning vectors, also possess the control elements necessary for transcription and translation, such as promoters and terminators.

[0092] [Amended according to Rule 26, 16.12.2024] As used herein, the term "control element" refers to the nucleic acid sequence necessary for expressing the polynucleotide encoding the truncated peptide of the present invention. The control sequence may be native (i.e., from the same gene) or exogenous (i.e., from different genes) to the polynucleotide encoding the polypeptide, or native or exogenous relative to each other. Such control elements include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control elements include promoters and transcription and translation termination signals.

[0093] [Amended according to Rule 26, 16.12.2024] As used in this text, an "integrative plasmid" means a DNA sequence containing an optional marker and at least one target gene expression cassette. Before being transformed into yeast, integrative plasmids are linearized, which allows their sequence to be inserted into regions of the yeast genome. Yeast integrative plasmids are genetic units that cannot replicate autonomously outside the chromosome and must be integrated into the yeast chromosome to replicate.

[0094] [Amended according to Rule 26 16.12.2024] As used herein, “yeast host cell” or “yeast cell” means any yeast host cell that is readily transformed, transfected, transduced, etc. by a nucleic acid construct or recombinant expression vector containing the polynucleotides of the present invention. Yeast host cells can be cells from the genera *Pichia*, *Candida*, *Hansenula*, *Kluyveromyces*, *Saccharomyces*, *Schizosaccharomyces*, or *Yarrowia*, such as *Kluyveromyces lactis*, *Saccharomyces carlsbergensis*, *Saccharomyces cerevisiae*, *Saccharomyces diastaticus*, *Saccharomyces douglasii*, *Saccharomyces kluyveri*, *Saccharomyces norbensis*, *Saccharomyces oviformis*, or *Yarrowia lipolytica*.

[0095] [Amended according to Rule 26 16.12.2024] As used herein, the term “expression” includes any step involved in the production of a polypeptide, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0096] [Amended according to Rule 26, 16.12.2024] As used herein, the term "protein docking model" refers to the use of CB Dock 2 for binding site analysis after potential receptors have been identified. CB Dock 2 combines indentation detection and the AutoDock Vina algorithm to predict potential binding sites by identifying indentations on the surface of the target protein. This method first identifies potential binding sites using geometric analysis and molecular surface features, and then evaluates the binding affinity of each binding site through molecular docking simulation.

[0097] [Amended according to Rule 26, 16.12.2024] As used herein, the term "RFDiffusion" is an advanced generative model that utilizes a diffusion process to generate samples in a high-dimensional space. The original structure is processed by progressively adding noise, and a trained denoising network is used to generate new peptides that meet specific functional requirements. This process involves not only the de novo generation of functionally defined peptide fragments but also the optimization of biocompatibility and physicochemical properties to ensure that the generated peptides possess biological activity.

[0098] [Amended according to Rule 26, December 16, 2024] Design and Screening Methods for Functional Protein Active Peptides

[0099] [Amended according to Rule 26, 16.12.2024] This invention provides a method for designing and screening functional protein active peptides, comprising the following steps:

[0100] [Amended according to Rule 26, 16.12.2024] 1) Recognition of functional protein binding receptors;

[0101] [Revised according to Rule 26, 16.12.2024] 2) Binding site analysis: Analyze potential binding sites on functional proteins that bind to receptors using protein docking models;

[0102] [Revised according to Rule 26, 16.12.2024] 3) Peptide design: Scaffold design based on binding sites using a diffusion model;

[0103] [Revised according to Rule 26, 16.12.2024] 4) Sequence generation: Use the ProteinMPNN model to generate the corresponding polypeptide amino acid sequence for the polypeptide backbone designed in step 3);

[0104] [Revised according to Rule 26, 16.12.2024] 5) Structure prediction: Perform three-dimensional structure prediction on the sequence generated in step 4);

[0105] [Revised according to Rule 26, 16.12.2024] 6) Result screening: Based on the structural prediction results of step 5), the first candidate polypeptide group is obtained through screening;

[0106] [Revised according to Rule 26, 16.12.2024] 7) Sequence alignment and candidate optimization: The first candidate polypeptide group obtained in step 6) is aligned with the known functional protein sequence to evaluate the similarity and difference between the candidate polypeptide sequence and the known functional protein sequence. According to the similarity, the target number of second candidate polypeptide groups with high similarity to the known functional protein sequence are selected.

[0107] [Revised according to Rule 26, 16.12.2024] 8) Experimental verification: Perform functional verification on the second candidate polypeptide group obtained in step 7). Based on the experimental data, rank the polypeptides according to their performance and obtain one or more target active polypeptides.

[0108] [Amended according to Rule 26, 16.12.2024] In some embodiments, in step 1), potential functional protein receptors are identified by searching a protein database (e.g., Uniprot) and conducting a literature search; the potential functional protein receptors are those that have been reported, predicted, or are predictable in prior art literature or protein databases.

[0109] [Amended according to Rule 26, 16.12.2024] In some embodiments, the protein docking model in step 2) uses the CB-Dock2 tool to analyze the binding sites of the identified receptors; preferably, the CB-Dock2 is based on cavity detection and the AutoDock Vina algorithm to identify potential binding sites on the protein surface.

[0110] [Amended according to Rule 26, 16.12.2024] In some embodiments, the potential binding sites identified by the protein docking model in step 2) are key sites for protein-protein interactions.

[0111] [Amended according to Rule 26 16.12.2024] In some embodiments, step 3) uses RFDiffusion to design 1000-3000 polypeptide backbones for each binding site; for example, about 1000, about 2000, or about 3000 polypeptide backbones are designed for each binding site.

[0112] [Revised according to Rule 26, 16.12.2024] In some embodiments, approximately 2000-3000 backbones are designed for all binding sites in step 3).

[0113] [Revised according to Rule 26, 16.12.2024] In some 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] [Amended according to Rule 26, 16.12.2024] In some embodiments, step 5) uses AlphaFold2 to predict the three-dimensional structure of the protein.

[0115] [Amended according to Rule 26 16.12.2024] In some embodiments, step 6) involves filtering based on the prediction score provided by AlphaFold2.

[0116] [Amended according to Rule 26, December 2024] In some implementations, the screening criteria include: plddt_binder>90 and / or plddt_target>90.

[0117] [Revised according to Rule 26, 16.12.2024] In some embodiments, the target quantity in step 7) is 50-200, for example, about 100.

[0118] [Amended according to Rule 26, 16.12.2024] In some embodiments, the experiment described in step 8) includes binding capacity experiments, cell experiments and / or animal experiments.

[0119] [Amended according to Rule 26, 16.12.2024] In some embodiments, the design and screening method for the functional protein active peptide may optionally include step 9) data analysis and feedback and step 10) iterative design.

[0120] [Amended according to Rule 26, 16.12.2024] In some embodiments, the data analysis and feedback includes in-depth analysis of experimental results to identify success and failure patterns. For success patterns: analyze the sequence and structural characteristics of peptides with excellent performance to find commonalities; for failure patterns: for peptides that did not achieve the expected results, analyze possible reasons, such as sequence instability, structural prediction deviations, etc. The analysis results are fed back into the model to adjust the model parameters.

[0121] [Amended according to Rule 26, 16.12.2024] In some embodiments, ProteinMPNN is optimized based on the characteristics of the successful peptide, the sequence generation strategy is optimized, and the model weights are adjusted.

[0122] [Amended according to Rule 26, 16.12.2024] In some implementations, AlphaFold2 is optimized based on the analysis results to improve the accuracy of structure prediction.

[0123] [Amended according to Rule 26, 16.12.2024] In some embodiments, the iterative design includes conducting a new round of peptide design, screening and validation based on the optimized model.

[0124] [Amended according to Rule 26, 16.12.2024] The design and screening method for functional protein active peptides provided by this invention can be widely used for the de novo design of various functional proteins, including but not limited to collagen, elastin, fibronectin, etc.

[0125] [Amended according to Rule 26, 16.12.2024] For example, the elastin peptide with Seq ID NO.1 in the applicant's prior patent CN118290568A was obtained using the functional protein active peptide design and screening method of the present invention. This elastin peptide has the ability to significantly promote the synthesis of its own elastin gene and fibronectin gene, and at the same time, it can significantly promote the synthesis of collagen IV and VII genes in the dermal-epidermal junction. UV resistance experiments have shown that this recombinant human elastin fragment can protect elastin, collagen I, IV and VII from UV damage and other excellent properties.

[0126] [Amended according to Rule 26 16.12.2024] For 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 functional protein active peptide design and screening method of the present invention. The above-mentioned fibronectin fragments have the effect of improving the skin barrier and increasing cell adhesion.

[0127] [Amended according to Rule 26, December 16, 2024] Design and Screening Methods for Type IV Collagen Cleavage Peptides

[0128] [Amended according to Rule 26, 16.12.2024] This 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 for integrin receptors; 3) Peptide design: using a diffusion model to design a backbone based on the binding sites; 4) Sequence generation: using a ProteinMPNN model to generate corresponding peptide amino acid sequences for the peptide backbone designed in step 3); 5) Structure prediction: performing three-dimensional structure prediction on the sequences generated in step 4); 6) Results Screening: Based on the structural prediction results of step 5), the first candidate polypeptide group is obtained through screening; 7) Sequence Alignment and Candidate Optimization: The first candidate polypeptide group obtained in step 6) is compared with known type IV collagen to evaluate the similarity and difference between the candidate polypeptide sequences and known type IV collagen. According to the similarity, the target number of second candidate polypeptide groups with high similarity to known functional protein sequences are obtained; 8) Experimental Validation: The second candidate polypeptide group obtained in step 7) is functionally validated. Based on the experimental data, the polypeptides are ranked according to their performance to obtain one or more active type IV collagen truncated peptides.

[0129] [Amended according to Rule 26, 16.12.2024] The screening method for type IV collagen active truncated peptides provided by this invention is as follows:

[0130] [Amended according to Rule 26 16.12.2024] 1) Receptor identification: Potential collagen IV receptors were identified through protein database searches and extensive literature searches.

[0131] [Amended according to Rule 26, December 16, 2024] 2) Binding Site Analysis: The CB-Dock 2 tool was used to analyze the binding sites of the identified integrin receptors. CB-Dock 2, based on cavity detection and the AutoDock Vina algorithm, can identify potential binding sites on the protein surface. Twelve hotspots were selected in the two pockets of collagen IV, which were considered key sites where collagen IV and integrins might interact.

[0132] [Amended according to Rule 26, December 16, 2024] 3) Peptide design: Novel peptide fragments were designed using RFDiffusion (diffusion model) around 12 identified hotspot regions. The design_ppi (protein-protein interaction) method of RFDiffusion was used, generating a total of 2000 peptide backbones for the hotspot regions.

[0133] [Revised according to Rule 26, December 16, 2024] 4) Using the ProteinMPNN (Protein Message Passing Neural Network) model, corresponding amino acid sequences were generated for the 2000 designed peptide backbones. When running ProteinMPNN, the parameter `--seqs_per_struct 4` was used to generate 4 different amino acid sequences for each peptide backbone. A total of 8000 candidate sequences were generated (2000 backbones × 4 sequences).

[0134] [Revised according to Rule 26, 16.12.2024] 5) The three-dimensional structure of the 8000 amino acid sequences generated was predicted using AlphaFold2.

[0135] [Revised according to Rule 26, December 2024] 6) Result Filtering:

[0136] [Revised according to Rule 26, December 16, 2024] In the obtained structural models, selection was performed based on the prediction scores provided by AlphaFold2, with particular attention paid to the PAE values ​​of protein-protein interaction regions in the Predicted Alignment Error (PAE) matrix. The selection criteria are as follows:

[0137] [Revised according to Rule 26, December 16, 2024] Selecting models with a PAE value of less than 5 indicates higher predictive reliability in the interaction region.

[0138] [Revised according to Rule 26, December 16, 2024] The root mean square deviation (RMSD) of the composite represents the spatial difference between the designed composite and the target structure; the smaller the value, the more similar the structures are.

[0139] [Revised according to Rule 26, December 16, 2024] The root mean square deviation after aligning the target and design components reflects the structural similarity between them.

[0140] [Revised according to Rule 26, December 16, 2024] The Predicted Alignment Error (PAE) of a composite is used to assess the accuracy of the relative positional information between atoms within the composite.

[0141] [Revised according to Rule 26, December 16, 2024] PAE values ​​for protein-protein interaction regions are used to ensure high-quality interaction predictions.

[0142] [Revised according to Rule 26, December 16, 2024] The PAE value of the target protein reflects the prediction error of the overall structure and ensures the stability of the target structure.

[0143] [Revised according to Rule 26, December 16, 2024] · plddt_binder>90: Predicted Local Distance Difference Test (PDD) of the binder, which reflects the overall reliability of the binder. The higher the value, the higher the reliability.

[0144] [Revised according to Rule 26, December 16, 2024] · plddt_target>90: Local deep learning score of the target protein, used to assess the reliability of the target structure.

[0145] [Revised according to Rule 26, December 16, 2024] · plddt_total>70: The total local deep learning score of the combined entity and the target, reflecting the reliability of the overall prediction.

[0146] [Revised according to Rule 26, December 2024] Based on the above screening criteria, 1030 high-quality structural models were selected from 8000 candidate sequences.

[0147] [Revised according to Rule 26, December 2024] 7) Sequence alignment and candidate optimization

[0148] [Revised according to Rule 26, December 16, 2024] Sequence alignment tools such as BLAST (Basic Local Alignment Search Tool) and Clustal Omega were used to evaluate the similarity and differences between candidate sequences and known sequences. One hundred candidate sequences with high similarity to known functional sequences were selected for subsequent experimental validation.

[0149] [Amended according to Rule 26, December 16, 2024] 8) High-throughput experimental verification: The selected sequences were functionally verified using a high-throughput experimental platform (such as Bota Freeway), the binding kinetics parameters of the peptides to the integrin receptor were determined, and the effectiveness of the peptides in biological function was evaluated 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] [Amended according to Rule 26, December 16, 2024] Type IV collagen cleavage peptide

[0151] [Amended according to Rule 26 16.12.2024] This 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 substitution, deletion or insertion of one or more amino acids based on SEQ ID NO: 1.

[0152] [Amended according to Rule 26, December 2024] GFPGQKGEKGQAGATGPKGLPGIPGAPGAPG(SEQ ID NO: 1)

[0153] [Amended according to Rule 26 16.12.2024] In some embodiments, the type IV collagen truncated peptide comprises the amino acid sequence shown in SEQ ID NO: 1.

[0154] [Amended according to Rule 26, 16.12.2024] In some embodiments, the type IV collagen truncated peptide consists of the amino acid sequence shown in SEQ ID NO: 1.

[0155] [Amended according to Rule 26, 16.12.2024] In some 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; even more preferably, the length is 31, 32, 33, 34, 35, 36, 37, 38, or 39 amino acids.

[0156] [Amended according to Rule 26 16.12.2024] In some embodiments, the type IV collagen truncated peptide has at least 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with natural type IV collagen (e.g., UniProt ID P29400), which has the amino acid sequence shown in SEQ ID NO: 21.

[0157] [Modified according to Rule 26, 16.12.2024] In some embodiments, the missing part occurs at both ends or in the middle of SEQ ID NO: 1.

[0158] [Amended according to Rule 26, 16.12.2024] In some embodiments, the absence is continuous or discontinuous.

[0159] [Amended according to Rule 26, 16.12.2024] In some embodiments, the deletion includes the consecutive deletion of up to 5, up to 4, up to 3, up to 2 amino acids at the N-terminus of SEQ ID NO: 1 and / or the consecutive deletion of up to 5, up to 4, up to 3, up to 2 amino acids at the C-terminus of SEQ ID NO: 1.

[0160] [Amended according to Rule 26, 16.12.2024] In some embodiments, the total number of amino acids deleted at the N-terminus and C-terminus of SEQ ID NO: 1 does not exceed 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0161] [Amended according to Rule 26, 16.12.2024] In some embodiments, the type IV collagen cleavage peptide may be modified or unmodified.

[0162] [Amended according to Rule 26, 16.12.2024] In some embodiments, the modification includes N-terminal modification, C-terminal modification, side chain modification, amino acid modification, backbone modification, etc.

[0163] [Amended according to Rule 26, 16.12.2024] In some embodiments, the modification includes cyclization, glycosylation, phosphorylation, N-methylation, myristylation and palmitoylation, polyethylene glycol (PEG) modification, etc.

[0164] [Revised according to Rule 26, 16.12.2024] In some embodiments, the cyclization modification is divided into sidechain-sidechain type, terminal-sidechain type, and terminal-terminal type.

[0165] [Amended according to Rule 26, 16.12.2024] In some embodiments, the cyclization modification forms collagen microspheres.

[0166] [Amended according to Rule 26, 16.12.2024] In some embodiments, variants of the type IV collagen truncated peptide have activity equivalent to or better than SEQ ID NO: 1.

[0167] [Amended according to Rule 26, December 16, 2024] Method for constructing recombinant bacteria

[0168] [Amended according to Rule 26, 16.12.2024] This invention provides a method for constructing yeast cells expressing type IV collagen truncated peptides, comprising introducing a recombinant expression vector into yeast cells, wherein the recombinant expression vector contains a polynucleotide encoding the collagen truncated peptide described herein. The recombinant expression vector may contain the nucleotide sequence shown in SEQ ID NO: 18. The yeast may be Pichia pastoris, preferably Pichia pastoris strain BG11.

[0169] [Amended according to Rule 26, 16.12.2024] The construction of the recombinant bacteria of the present invention includes the following steps: 1) design of collagen Col4A5 truncated peptide synthesis gene, 2) construction of expression cassette, 3) construction of integrative plasmid, and 4) transformation of yeast.

[0170] [Amended according to Rule 26, 16.12.2024] The collagen Col4A5 truncated peptide synthesis gene of the present invention encodes a signal peptide at the N-terminus and a truncated peptide shown in SEQ ID NO: 1 of the purified tag at the C-terminus.

[0171] [Amended according to Rule 26, 16.12.2024] In some embodiments, the signal peptide is a hybrid signal peptide composed of the Ost1 secreted signal peptide of Saccharomyces cerevisiae and the pro signal peptide of 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] [Amended according to Rule 26, 16.12.2024] In some embodiments, the purification tag is selected from HIS-Tag, GST-Tag, MBP-Tag, and NusA-Tag.

[0173] [Amended according to Rule 26, 16.12.2024] In some embodiments, the synthetic gene is optimized using codon optimization techniques. Preferably, the codon optimization techniques include the "Codon Optimization Tool" available on the website of Integrated DNA Technologies, Inc.

[0174] [Amended according to Rule 26, 16.12.2024] The collagen Col4A5 truncated peptide expression cassette of the present invention includes a synthetic gene, a promoter, and a terminator.

[0175] [Amended according to Rule 26, 16.12.2024] In some embodiments, the promoter is a methanol-inducible promoter; preferably, the methanol-inducible promoter is derived from the AOX1 gene promoter, the sequence of which is shown in SEQ ID NO: 4.

[0176] [Amended according to Rule 26, 16.12.2024] In some embodiments, the terminator is derived from the AOX1 gene transcription terminator, the sequence of which is shown in SEQ ID NO: 5.

[0177] [Amended according to Rule 26, 16.12.2024] The collagen Col4A5 truncated peptide expression cassette of the present invention is integrated into the yeast genome via an integrative plasmid. The integrative plasmid includes the expression cassette and a plasmid backbone.

[0178] [Amended according to Rule 26, 16.12.2024] In some embodiments, the backbone of the integrative plasmid includes: a 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] [Amended according to Rule 26 16.12.2024] In some embodiments, the backbone of the integrative plasmid includes the nucleotide sequence shown in SEQ ID NO: 9.

[0180] [Amended according to Rule 26, 16.12.2024] In some embodiments, the integrative plasmid, as shown in FIG1, comprises the nucleotide sequence shown in SEQ ID NO: 18.

[0181] [Amended according to Rule 26, 16.12.2024] The integrative plasmid of the present invention is linearized and then transformed into a yeast strain and integrated into the yeast genome.

[0182] [Amended according to Rule 26 16.12.2024] In some embodiments, the integrative plasmid is linearized using the PmeI restriction enzyme, which cuts at the middle position of the methanol-induced promoter sequence of the AOX1 gene.

[0183] [Revised according to Rule 26, December 16, 2024] Recombinant microbial fermentation method

[0184] [Amended according to Rule 26, 16.12.2024] This invention provides a method for producing type IV collagen truncated peptides by fermentation of yeast transformed with an integrative plasmid.

[0185] [Amended according to Rule 26 16.12.2024] In some embodiments, seed culture is performed prior to fermentation culture.

[0186] [Amended according to Rule 26, 16.12.2024] In some embodiments, the fermentation culture medium includes the basal culture medium of Table 1 and the additives of Table 2.

[0187] [Amended according to Rule 26, 16.12.2024] In some embodiments, the seed is added to the fermentation medium, the initial OD of the fermentation culture is 0.2-0.4, the fermentation culture is 20-26h, and glycerol is added at the beginning, the initial addition amount is 23-33ml / kg / h, and the subsequent addition amount of glycerol is controlled according to the dissolved oxygen to maintain the dissolved oxygen at about 20%.

[0188] [Amended according to Rule 26, 16.12.2024] In some embodiments, the seed is added to the above-mentioned fermentation medium with an initial OD of 0.3. After fermentation for 22-24 hours, glycerol is added at an initial rate of 28 ml / kg / h. The amount of glycerol added is then controlled according to dissolved oxygen levels to maintain dissolved oxygen at around 20%.

[0189] [Amended according to Rule 26, 16.12.2024] In some embodiments, the cells are cultured until the wet weight reaches 330-380 g / L, and then switched to methanol for induction. The initial addition of methanol is 0.4-0.6 ml / kg / h, and the methanol addition rate is subsequently adjusted according to dissolved oxygen to maintain dissolved oxygen at around 40%. The induction time is 150-190 h.

[0190] [Amended according to Rule 26, 16.12.2024] In some embodiments, the cells are cultured until the wet weight reaches 330-380 g / L, and then switched to methanol for induction. The initial amount of methanol added is 0.5 ml / kg / h, and the methanol addition rate is subsequently adjusted according to dissolved oxygen to maintain dissolved oxygen at around 40%. The induction time is 170 h.

[0191] [Amended according to Rule 26, 16.12.2024] This invention provides a method for separation and purification after fermentation culture.

[0192] [Amended according to Rule 26, 16.12.2024] In some embodiments, the separation includes taking the cultured bacterial solution, centrifuging the bacterial solution in a low-temperature centrifuge, and retaining the supernatant.

[0193] [Amended according to Rule 26 16.12.2024] In some embodiments, the separation includes filtering the supernatant with a 0.45 μm filter membrane.

[0194] [Amended according to Rule 26, 16.12.2024] In some embodiments, the purification method includes affinity chromatography column chromatography.

[0195] [Amended according to Rule 26, 16.12.2024] In some embodiments, the chromatography includes: flowing the sample through a Ni column at a rate of 1 ml / min; equilibrating the column with Buffer A (20 mM PB, pH 7.5); eluting with 20 mM, 300 mM, and 500 mM imidazole; and performing SDS-PAGE gel electrophoresis on the eluted sample to analyze whether the target protein is present.

[0196] [Amended according to Rule 26, December 16, 2024] This application also provides the following implementation scheme:

[0197] [Amended according to Rule 26, 16.12.2024] 1. A polypeptide, characterized in that it comprises the amino acid sequence shown in SEQ ID NO: 1 or a variant amino acid sequence obtained by substitution, deletion or insertion of one or more amino acids based on SEQ ID NO: 1; the polypeptide does not contain a full-length type IV collagen sequence.

[0198] [Amended according to Rule 26, 16.12.2024] 2. The polypeptide according to Embodiment 1 is a truncated peptide of type IV collagen; preferably, the type IV collagen has the amino acid sequence shown in SEQ ID NO:21.

[0199] [Amended according to Rule 26, 16.12.2024] 3. The polypeptide according to Embodiment 1, comprising the amino acid sequence shown in SEQ ID NO: 1 and having a length of 31-50 amino acids; preferably, having a length of 31-45 amino acids; more preferably, having a length of 31-40 amino acids.

[0200] [Amended according to Rule 26, 16.12.2024] 4. The polypeptide according to Embodiment 1, wherein the deletion includes 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; preferably, the total number of amino acids deleted at the N-terminus and C-terminus of SEQ ID NO: 1 does not exceed 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0201] [Amended according to Rule 26, 16.12.2024] 5. The polypeptide according to Embodiment 1, wherein the variant has activity equivalent to or better than SEQ ID NO: 1; said activity includes mitochondrial protection, skin tissue morphology protection and / or promotion of expression of endogenous anti-aging related proteins; preferably, said anti-aging related proteins include type IV collagen, type VII collagen, type XVII collagen and / or laminin 5.

[0202] [Amended according to Rule 26 16.12.2024] 6. A polynucleotide encoding a polypeptide as described in any one of embodiments 1-5.

[0203] [Amended according to Rule 26 16.12.2024] 7. The polynucleotide according to Embodiment 6 further comprises a sequence encoding a purification tag and / or a signal peptide sequence;

[0204] [Amended according to Rule 26, 16.12.2024] Preferably, the signal peptide is a hybrid signal peptide composed of Ost1 secretory signal peptide and pro signal peptide;

[0205] [Amended according to Rule 26, 16.12.2024] Preferably, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2;

[0206] [Amended according to Rule 26, 16.12.2024] Preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 3 or a degenerate sequence thereof.

[0207] [Amended according to Rule 26, 16.12.2024] 8. A vector comprising the polynucleotide described in Embodiment 6 or 7;

[0208] [Amended according to Rule 26, 16.12.2024] Preferably, the vector is an integrative plasmid;

[0209] [Amended according to Rule 26, 16.12.2024] Preferably, the integrative plasmid further includes a methanol-inducible promoter of the AOX1 gene and / or a transcription terminator of the AOX1 gene;

[0210] [Amended according to Rule 26, 16.12.2024] Preferably, the backbone sequence of the integrative plasmid includes a G418 resistance gene cassette, an ampicillin resistance gene cassette, and / or a Col E1 replicon;

[0211] [Amended according to Rule 26, 16.12.2024] Preferably, 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%, or 99% identity with SEQ ID NO: 9;

[0212] [Amended according to Rule 26, 16.12.2024] Preferably, 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.

[0213] [Amended according to Rule 26 16.12.2024] 9. A cell comprising the polynucleotide of embodiment 6 or 7 or the carrier of embodiment 8; preferably, the cell is a eukaryotic cell; more preferably, the eukaryotic cell is a yeast host cell.

[0214] [Amended according to Rule 26 16.12.2024] 10. A composition, characterized in that it comprises the polypeptide described in any one of embodiments 1-5; preferably, the composition is formulated as a dosage form suitable for administration by means selected from oral, topical and injection methods.

[0215] [Amended according to Rule 26, 16.12.2024] 11. Use of the polypeptide described in any one of Embodiments 1-5 or the composition described in Embodiment 10 in the preparation of cosmetics, health products, food additives or pharmaceuticals.

[0216] [Amended according to Rule 26, 16.12.2024] 12. A method for producing the polypeptide according to any one of embodiments 1-5, characterized in that it includes the following steps: fermenting and culturing the cells according to embodiment 9, and separating and purifying the polypeptide from the culture; preferably, the cells are yeast cells.

[0217] [Revised according to Rule 26, 16.12.2024] 13. The method according to Implementation Scheme 12, wherein methanol induction is performed during yeast fermentation expression.

[0218] [Amended according to Rule 26, December 2024] 14. A method for designing and screening functional protein active peptides, characterized by comprising the following steps:

[0219] [Amended according to Rule 26, 16.12.2024] 1) Recognition of functional protein binding receptors;

[0220] [Revised according to Rule 26, 16.12.2024] 2) Binding site analysis: Analyze potential binding sites on functional proteins that bind to receptors using protein docking models;

[0221] [Revised according to Rule 26, 16.12.2024] 3) Peptide design: Scaffold design based on binding sites using a diffusion model;

[0222] [Revised according to Rule 26, 16.12.2024] 4) Sequence generation: Use the ProteinMPNN model to generate the corresponding polypeptide amino acid sequence for the polypeptide backbone designed in step 3);

[0223] [Revised according to Rule 26, 16.12.2024] 5) Structure prediction: Perform three-dimensional structure prediction on the sequence generated in step 4);

[0224] [Revised according to Rule 26, 16.12.2024] 6) Result screening: Based on the structural prediction results of step 5), the first candidate polypeptide group is obtained through screening;

[0225] [Revised according to Rule 26, 16.12.2024] 7) Sequence alignment and candidate optimization: The first candidate polypeptide group obtained in step 6) is aligned with the known functional protein sequence to evaluate the similarity and difference between the candidate polypeptide sequence and the known functional protein sequence. According to the similarity, the target number of second candidate polypeptide groups with high similarity to the known functional protein sequence are selected.

[0226] [Revised according to Rule 26, 16.12.2024] 8) Experimental verification: Perform functional verification on the second candidate polypeptide group obtained in step 7). Based on the experimental data, rank the polypeptides according to their performance and obtain one or more target active polypeptides.

[0227] [Revised according to Rule 26, 16.12.2024] 15. According to the method described in Implementation Scheme 14, in step 1), potential functional protein receptors are identified by searching protein databases and literature.

[0228] [Revised according to Rule 26, 16.12.2024] 16. According to the method described in Implementation Scheme 14, 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 to identify potential binding sites on the protein surface.

[0229] [Revised according to Rule 26, 16.12.2024] 17. According to the method described in Implementation Scheme 14, in step 3), 1000-3000 polypeptide backbones are designed for each binding site.

[0230] [Revised according to Rule 26, 16.12.2024] 18. According to the method described in Implementation Scheme 14, in step 4), 2-6 polypeptide sequences are generated for each polypeptide backbone; preferably, 4 polypeptide sequences are generated for each polypeptide backbone.

[0231] [Revised according to Rule 26, 16.12.2024] 19. According to the method described in Implementation Scheme 14, in step 5), AlphaFold2 is used to predict the three-dimensional structure of the protein.

[0232] [Revised according to Rule 26, 16.12.2024] 20. According to the method described in Implementation Scheme 14, in step 6), the selection is performed based on the prediction score provided by AlphaFold2, and the selection criteria include: And / or plddt_target>90.

[0233] [Revised according to Rule 26, 16.12.2024] 21. According to the method of implementation scheme 14, the experiment in step 8) includes binding capacity experiment, cell experiment and / or animal experiment.

[0234] [Amended according to Rule 26, 16.12.2024] 22. The method according to any one of Implementation Schemes 14-21, wherein the functional protein is type IV collagen; the experiments in step 8) include experiments on binding ability to integrin receptors, cell adhesion and migration experiments.

[0235] [Revised according to Rule 26, 16.12.2024] 23. The active peptide obtained by screening according to the method of implementation scheme 22 is as described in any one of implementation schemes 1-5.

[0236] [Amended according to Rule 26, 16.12.2024] 24. Application of the method described in any one of Implementation Schemes 14-23 in cosmetic ingredient development, drug development and / or basic research.

[0237] [Amended according to Rule 26, December 2024] Example

[0238] [Amended according to Rule 26, 16.12.2024] The invention will be more readily understood by referring to the following examples, which are only used to illustrate certain aspects and embodiments of the invention and are not intended to limit the invention.

[0239] [Amended according to Rule 26, 16.12.2024] Unless otherwise stated, all reagents used in this embodiment are commercially available or conventional materials.

[0240] [Amended according to Rule 26, December 16, 2024] Example 1: Screening of Type IV Collagen Cleavage Peptides

[0241] [Amended according to Rule 26, December 16, 2024] Using bioinformatics methods, potential receptors for type IV collagen are identified. A protein docking model is applied to identify potential binding peptide sites. A diffusion pooling model is used to design peptide fragments, which are then compared with the amino acid sequences of six different chains of type IV collagen to screen for potential peptides. These fragments are then validated through wet-lab experiments to screen for target peptides. Specifically, the method includes the following steps:

[0242] [Amended according to Rule 26 16.12.2024] 1) Receptor identification: Identify potential type IV collagen receptors through protein database searches (such as Uniprot) and extensive literature searches.

[0243] [Amended according to Rule 26, December 16, 2024] 2) Binding Site Analysis: The CB-Dock 2 tool was used to analyze the binding sites of the identified integrin receptors. CB-Dock 2, based on cavity detection and the AutoDock Vina algorithm, can identify potential binding sites on the protein surface. Specifically, 12 hotspots were selected in the two pockets of type IV collagen. These hotspots are considered key sites where type IV collagen and integrins may interact. In-depth analysis of these hotspots provided important structural information for subsequent peptide design.

[0244] [Amended according to Rule 26, December 16, 2024] 3) Peptide design:

[0245] [Amended according to Rule 26, December 16, 2024] Novel peptide fragment designs were performed around 12 identified hotspot regions using RFDiffusion (diffusion model). RFDiffusion is a diffusion-based generative model capable of generating peptide structures that meet specific functional requirements in a high-dimensional space. Specifically, the design_ppi (protein-protein interaction) method of RFDiffusion was used to generate a total of 2000 peptide backbones for the 12 hotspot regions.

[0246] [Revised according to Rule 26, December 16, 2024] 4) Sequence generation:

[0247] [Revised according to Rule 26, December 16, 2024] The ProteinMPNN (Protein Message Passing Neural Network) model was used to generate corresponding amino acid sequences for the generated polypeptide backbone. The parameter `--seqs_per_struct 4` was used when running ProteinMPNN, meaning that four different amino acid sequences were generated for each polypeptide backbone. This resulted in a total of 8000 candidate sequences (2000 backbones × 4 sequences).

[0248] [Revised according to Rule 26, December 16, 2024] Features and workflow of ProteinMPNN:

[0249] [Revised according to Rule 26, December 2024] • Graph Neural Network: Represents the polypeptide backbone as a graph structure, where nodes represent amino acids and edges represent interactions.

[0250] [Revised according to Rule 26, December 16, 2024] • Sequence optimization: Based on the backbone structure, the model predicts the most suitable amino acid sequence and optimizes the sequence to improve stability and functionality.

[0251] [Revised according to Rule 26, December 16, 2024] Parallel generation: Generates multiple sequences at once, increasing the diversity of candidate sequences.

[0252] [Amended according to Rule 26, December 2024] 5) Structure prediction: The generated 8000 amino acid sequences were used to predict their three-dimensional structure using AlphaFold2. AlphaFold2 is a deep learning-based protein structure prediction model that can accurately predict the three-dimensional folding structure of proteins. This invention uses AlphaFold2's initial guess mode to improve prediction efficiency.

[0253] [Revised according to Rule 26, December 16, 2024] How AlphaFold2 works:

[0254] [Amended according to Rule 26, December 16, 2024] • Multiple sequence alignment (MSA): Constructing multiple sequence alignments of the target sequence using evolutionary information. • Neural network prediction: Predicting structural information such as distance maps and angular distributions using deep neural networks.

[0255] [Revised according to Rule 26, December 16, 2024] • Structure Reconstruction: Reconstruct the three-dimensional structure of the protein based on the predicted structural information.

[0256] [Revised according to Rule 26, December 2024] 6) Result Filtering:

[0257] [Revised according to Rule 26, December 16, 2024] In the obtained structural models, selection was performed based on the prediction scores provided by AlphaFold2, with particular attention paid to the PAE values ​​of protein-protein interaction regions in the Predicted Alignment Error (PAE) matrix. The selection criteria are as follows:

[0258] [Revised according to Rule 26, December 16, 2024] Selecting models with a PAE value of less than 5 indicates higher predictive reliability in the interaction region.

[0259] [Revised according to Rule 26, December 16, 2024] The root mean square deviation (RMSD) of the composite represents the spatial difference between the designed composite and the target structure; the smaller the value, the more similar the structures are.

[0260] [Revised according to Rule 26, December 16, 2024] The root mean square deviation after aligning the target and design components reflects the structural similarity between them.

[0261] [Revised according to Rule 26, December 16, 2024] The Predicted Alignment Error (PAE) of a composite is used to assess the accuracy of the relative positional information between atoms within the composite.

[0262] [Revised according to Rule 26, December 16, 2024] PAE values ​​for protein-protein interaction regions are used to ensure high-quality interaction predictions.

[0263] [Revised according to Rule 26, December 16, 2024] The PAE value of the target protein reflects the prediction error of the overall structure and ensures the stability of the target structure.

[0264] [Revised according to Rule 26, December 16, 2024] · plddt_binder>90: Predicted Local Distance Difference Test (PDD) of the binder, which reflects the overall reliability of the binder. The higher the value, the higher the reliability.

[0265] [Revised according to Rule 26, December 16, 2024] · plddt_target>90: Local deep learning score of the target protein, used to assess the reliability of the target structure.

[0266] [Revised according to Rule 26, December 16, 2024] · plddt_total>70: The total local deep learning score of the combined entity and the target, reflecting the reliability of the overall prediction.

[0267] [Revised according to Rule 26, December 2024] Based on the above screening criteria, 1030 high-quality structural models were selected from 8000 candidate sequences.

[0268] [Revised according to Rule 26, December 16, 2024] 7) Sequence alignment and candidate optimization:

[0269] [Amended according to Rule 26, December 16, 2024] The 1030 selected candidate sequences 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 similarity and differences between the candidate sequences and known sequences.

[0270] [Revised according to Rule 26, December 16, 2024] Comparison process:

[0271] [Revised according to Rule 26, December 16, 2024] · BLAST alignment: The candidate sequence is compared with the known sequences in the database to obtain a similarity score.

[0272] [Amended according to Rule 26, December 16, 2024] • Multiple sequence alignment: Clustal Omega is used to align multiple sequences to identify conserved regions and variant sites.

[0273] [Revised according to Rule 26, December 2024] • Screening criteria: Select candidate sequences that have high similarity to known functional sequences.

[0274] [Revised according to Rule 26, 16.12.2024] Through comparative analysis, 100 candidate sequences with high similarity to known functional sequences were selected for subsequent experimental verification.

[0275] [Revised according to Rule 26, December 2024] 8) Experimental verification:

[0276] [Amended according to Rule 26, December 16, 2024] The company’s high-throughput experimental platform, Bota Freeway, was used to express and validate the functions of 100 selected candidate peptides.

[0277] [Revised according to Rule 26, December 2024] Experimental Procedure:

[0278] [Revised according to Rule 26, 16.12.2024] • Peptide synthesis: Synthesize 100 candidate peptides, ensuring that the purity and quality meet the experimental requirements.

[0279] [Amended according to Rule 26, 16.12.2024] • Binding experiment: Surface plasmon resonance (SPR) technology was used to determine the binding kinetic parameters (such as binding constant and dissociation constant) of the peptide and the integrin receptor.

[0280] [Amended according to Rule 26, 16.12.2024] • Functional assessment: Evaluate the effectiveness of the peptide in biological function through cell experiments (such as cell adhesion and migration experiments).

[0281] [Amended according to Rule 26, December 16, 2024] • Data collection: Record experimental data for each peptide, including binding affinity, functional activity, etc.

[0282] [Revised according to Rule 26, December 2024] Result Analysis:

[0283] [Amended according to Rule 26, December 16, 2024] • Success rate: Among 100 candidate peptides, a subset of peptides were found to exhibit the expected high affinity and biological activity.

[0284] [Amended according to Rule 26, December 16, 2024] • Performance Ranking: Based on experimental data, the peptides were ranked according to their performance, and a 31-amino acid fragment of the 5α chain of type IV collagen (SEQ ID NO: 1, GFPGQKGEKGQAGATGPKGLPGIPGAPGAPG) was selected as the best performing peptide for further research.

[0285] [Revised according to Rule 26, December 2024] Example 2: Synthesis, isolation, and purification of the original protein Col4A5 truncated peptide

[0286] [Amended according to Rule 26, December 16, 2024] 2.1 Construction of Collagen Col4A5 Truncation Peptide Integral Plasmid Expression System

[0287] [Amended according to Rule 26, December 16, 2024] Collagen Col4A5 truncated peptide synthesis gene

[0288] [Amended according to Rule 26, 16.12.2024] Collagen Col4A5 truncated peptide (SEQ ID NO:1, GFPGQKGEKGQAGATGPKGLPGIPGAPGAPG) is a fragment (amino acids 509-539) of human collagen Col4A5 (UniProt ID P29400), obtained by screening according to the method described in Example 1. For ease of purification, a His tag (-GSSHHHHHH) was attached to the C-terminus. This peptide was designed for secretory expression by 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 from the pre-pro signal peptide of mating factor α, was placed upstream of the collagen Col4A5 truncated peptide sequence to guide secretion. To improve expression efficiency, codon optimization techniques were used to design and optimize the product gene sequence. Specifically, the nucleotide sequences encoding the signal peptide and the collagen Col4A5 truncated peptide with a His tag at the C-terminus were codon optimized using the "Codon Optimization Tool" from Integrated DNA Technologies, Inc.'s website. The codon-optimized gene sequence (SEQ ID NO: 3) was subsequently synthesized by BGI TECH SOLUTIONS (BEIJING LIUHE) CO., LIMITED.

[0289] [Amended according to Rule 26, December 16, 2024] Collagen Col4A5 truncated peptide expression cassette

[0290] [Amended according to Rule 26, 16.12.2024] The collagen Col4A5 truncated peptide expression cassette comprises 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 previously described (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). An integrative plasmid was used to integrate the collagen Col4A5 truncated peptide expression cassette into the yeast genome.

[0291] [Revised from Rule 26, December 16, 2024] Construction of integrative plasmids

[0292] [Amended according to Rule 26, December 16, 2024] The backbone of the integrative plasmid comprises: a 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 BGI TECH SOLUTIONS (BEIJING LIUHE) CO., LIMITED (SEQ ID NO: 9).

[0293] [Amended according to Rule 26 16.12.2024] 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).

[0294] [Amended 16.12.2024 according to Rule 26] Genomic DNA of BG11 strain (PS10011) 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-induced promoter sequence of the AOX1 gene (SEQ ID NO: 4) was amplified using primers Pro-F and Pro-R (SEQ ID NO: 12 and SEQ ID NO: 13).

[0295] [Amended according to Rule 26, December 2024] The 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 BGI TECH SOLUTIONS (BEIJING LIUHE) CO., LIMITED (SEQ ID NO: 9) (SEQ ID NO: 3).

[0296] [Amended according to Rule 26 16.12.2024] Using genomic DNA of BioGrammatics Inc. strain BG11 (PS10011) as a template, the transcription terminator of the AOX1 gene (SEQ ID NO: 5) was amplified using primers Ter-F and Ter-R (SEQ ID NO: 16 and SEQ ID NO: 17).

[0297] [Amended according to Rule 26, December 16, 2024] The amplification products were ligated using NEB's NEBuilder HiFi DNA Assembly Premix Kit, and the ligation products were 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).

[0298] [Revised according to Rule 26, December 16, 2024] Construction of a collagen Col4A5 truncated peptide expression strain

[0299] [Amended 16.12.2024 according to Rule 26] The integrative 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 cleaves at the middle position of the methanol-inducible promoter sequence of the AOX1 gene.

[0300] [Amended 16.12.2024 according to Rule 26] BG11 strain (PS 10011, BioGrammatics Inc.) was transformed by electroporation using 100 ng of linearized plasmid. Competent cells were prepared and electroporation was performed in accordance with the protocol in Methods in Enzymology, 2021 Vol 660, 53-80.

[0301] [Amended 16.12.2024 according to Rule 26] Transformants were screened on YPD solid medium containing 1 g / L G418.

[0302] [Amended according to Rule 26, December 16, 2024] Integration of the integrative plasmid into the methanol-inducible promoter site of 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 methanol-inducible promoter of the AOX1 gene, 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 was expected to produce a 1.9 kb PCR product (Figure 2). The presence of the collagen expression vector was confirmed by Sanger sequencing of the PCR product. The colony PCR procedure was derived from "Protein Expression in Pichia pastoris," Enzymological Methods, Vol. 660, 2021, pp. 53-80. The validated transformant strain was named Strain-Col4A5pep.

[0303] [Revised according to Rule 26, December 16, 2024] Collagen Col4A5 truncated peptide yeast fermentation process

[0304] [Revised according to Rule 26, 16.12.2024] The composition of the fermentation medium is shown in Tables 1-2. It is sterilized at 121°C for 30 minutes, cooled to 28°C for later use, and PTM1 and HMP are added after cooling.

[0305] [Revised according to Rule 26, December 16, 2024] Table 1: Composition of Fermentation Medium

[0306] [Revised according to Rule 26, December 16, 2024] Table 2 PTM1 Formulation

[0307] [Revised from Rule 26, December 2024] The single-clone strain was added to 50 ml of LYPD medium and cultured overnight at 28°C and 250 rpm. The next day, the seed culture from the primary shake flask was transferred to 100 ml of LYPD medium, with an OD of 0.5, and cultured at 28°C and 250 rpm for 24 hours as the incubation seed. The incubation seed was added to the above fermentation medium, with an initial OD of 0.3, and fermented for 22-24 hours. Glycerol was then added initially at 28 ml / kg / h, and the amount of glycerol was subsequently adjusted according to dissolved oxygen levels to maintain dissolved oxygen at around 20%. The culture was continued until the cell wet weight reached 330-380 g / L, then methanol was switched for induction. The initial amount of methanol was 0.5 ml / kg / h, and the methanol addition rate was adjusted according to dissolved oxygen levels to maintain dissolved oxygen at around 40%. The induction time was 170 hours. The yield of Col4A5 at different fermentation time points was detected using SDS-PAGE and BCA, and the results are shown in Figures 3 and 4.

[0308] [Amended according to Rule 26, December 16, 2024] Purification of Collagen Col4A5 truncated peptide

[0309] [Revised according to Rule 26, 16.12.2024] Take the cultured bacterial solution, centrifuge it in a low-temperature centrifuge, and retain the supernatant.

[0310] [Amended according to Rule 26, December 2024] The supernatant obtained above was filtered through a 0.45 μm filter membrane, and then purified by protein purification using a 5 ml affinity chromatography column. The steps are as follows:

[0311] [Amended according to Rule 26, 16.12.2024] 1) Flow the sample through the Ni column at a rate of 1 ml / min;

[0312] [Revised according to Rule 26, 16.12.2024] 2) Equilibrate the column with Buffer A (20mM PB, pH 7.5);

[0313] [Amended according to Rule 26 16.12.2024] 3) Elute with 20mM, 300mM, and 500mM imidazole.

[0314] [Revised according to Rule 26, 16.12.2024] 4) Perform SDS-PAGE gel analysis on the eluted samples to determine if the target protein is present.

[0315] [Amended according to Rule 26, December 2024] Example 3: Protective effect of collagen Col4A5 truncated peptide on mitochondria.

[0316] [Amended according to Rule 26, 16.12.2024] The protective effect of the Col4A5 truncated peptide on mitochondria was verified by mitochondrial membrane potential (ΔΨM) analysis.

[0317] [Revised according to Rule 26, December 2024] Experimental Grouping:

[0318] [Revised according to Rule 26, December 2024] Table 3: Mitochondrial membrane potential (ΔΨM) analysis grouping

[0319] [Revised according to Rule 26, December 16, 2024] Experimental Procedure

[0320] [Amended according to Rule 26, December 2024] Human primary skin fibroblasts (HDFs) (lifeline, #FC-0024) were expanded and cultured using fibroblast culture medium under the conditions of 37°C, 95% humidity, and 5% CO2. When the cells reached 80-90% confluence, they were digested with trypsin and transferred to 96-well plates. When the cells reached 90% confluence in the 96-well plates, they were treated with 10 J / cm²... 2 Cells were irradiated with a UVA lamp of high intensity. Immediately after UVA irradiation, HDFs were treated according to experimental groups and incubated for another 24 hours. The cell culture medium was removed, and 2 μM (final concentration) of JC-1 dye was added. Cells were incubated at 37°C and 5% CO2 for 20 minutes. The dye solution was then removed, and the cells were washed twice with buffer. JC-1 fluorescence of the test cultures (including the control group) was analyzed immediately. For quantitative methods, red fluorescence (excitation 568 nm / emission 590 nm) and green fluorescence (excitation 485 nm / emission 535 nm) were measured using a multi-plate reader. The ratio of red fluorescence to green fluorescence was then determined. Simultaneously, fluorescence images of live cells on coverslips were captured.

[0321] [Amended according to Rule 26, December 16, 2024] Note: In apoptotic and dead cells, the dye will appear green in monomeric form; while in living, 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" and accumulate in the mitochondria. J-aggregates form a broad excitation spectrum and have a maximum emission peak at approximately 590 nm. When the JC-1 dye is present in low mitochondrial concentrations or at low ΔΨM, it exists in monomeric form with an emission wavelength of 535 nm.

[0322] [Revised according to Rule 26, December 16, 2024] Result

[0323] [Revised according to Rule 26, December 2024] The results are shown in Figure 5. The results show that, compared to the blank control group (BC), the negative control group (10 J / cm²)... 2 A significant decrease in mitochondrial membrane potential due to UVA (UVA) ### p < 0.001 indicates that the model of UVA radiation-induced mitochondrial damage in fibroblasts was successfully established. Compared with the negative control group, the mitochondrial membrane potential of fibroblasts treated with 100 ppm recombinant type IV collagen truncated peptide was significantly increased (***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.

[0324] [Amended according to Rule 26, December 16, 2024] Example 4: The promoting effect of collagen Col4A5 truncated peptide on the expression of aging-related proteins.

[0325] [Amended according to Rule 26, 16.12.2024] The promoting effect of Col4A5 truncated peptide on the expression of aging-related proteins was analyzed by in vitro skin experiments.

[0326] [Revised according to Rule 26, December 2024] Experimental Grouping:

[0327] [Revised according to Rule 26, December 16, 2024] Table 4 Grouping of Ex vivo skin experiments

[0328] [Revised according to Rule 26, December 2024] Experimental Procedure:

[0329] [Revised according to Detailed Rule 26, December 16, 2024] 1) Organizational Processing

[0330] [Amended according to Rule 26, December 16, 2024] Freshly obtained skin tissue (Guangdong Boxi Biotechnology Co., Ltd.) was immersed in 75% alcohol for 30 seconds, and then washed three times with sterile PBS buffer. After the washing was completed, the skin was cut into 24±2 mm pieces. 2 The tissue block, with the epidermis facing up and the dermis facing down, was placed in a culture mold. The culture mold was then transferred into a 6-well plate, and 3.7 mL of culture medium was added to each well. The plate was then incubated at 37°C in a 5% CO2 incubator, with the medium changed daily.

[0331] [Amended according to Rule 26, December 16, 2024] 2) Administration

[0332] [Revised according to Rule 26, December 2024] After one day of culture of ex vivo skin tissue, drugs and irradiation were administered according to the experimental grouping and corresponding treatment conditions. For the first four days, UVA irradiation was performed daily (the irradiation dose was UVA (30 J / cm²)).2 ) and UVB (50mJ / cm 2 Irradiation (approximately 35 minutes) is performed. After irradiation, the test substance is applied to the tissue surface (the remaining test substance must be removed before applying the test substance the next day), and the culture medium is replaced with fresh medium. After 4 consecutive days, only drug administration is performed, that is, the test substance is applied to the tissue surface daily (the remaining test substance must be removed before applying the test substance the next day), for 3 consecutive days.

[0333] [Revised according to Rule 26, December 16, 2024] 3) Organizational morphology test

[0334] [Amended according to Rule 26, 16.12.2024] Skin tissue for tissue morphology testing was taken, fixed with 4% paraformaldehyde, and after 24 hours of fixation, H&E staining was performed, and the tissue was photographed and observed under a microscope. The images were collected and analyzed.

[0335] [Revised according to Rule 26, December 16, 2024] 4) Immunofluorescence detection

[0336] [Amended according to Rule 26, December 16, 2024] After drug administration, skin tissue was fixed with 4% paraformaldehyde, then embedded and sectioned. Paraffin sections were placed in a 70°C slide oven for 4 hours. Sections were then immersed in xylene for 10 minutes, the xylene was replaced, and the sections were immersed for another 10 minutes, followed by immersion in anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, and 75% ethanol for 5 minutes. The sections were washed three times with PBS buffer, 5 minutes each time. Paraffin sections were then placed in 0.01M sodium citrate antigen retrieval solution and autoclaved. After cooling, the sections were removed. The sections were washed three times with PBS buffer, 5 minutes each time. One drop of 3% H2O2 was added to each section, and the sections were incubated at room temperature for 30 minutes to block endogenous peroxidase activity. The sections were washed three times with PBS buffer, 5 minutes each time. Serum homologous to the secondary antibody was added and the sections were blocked at 37°C for 60 minutes without rinsing. Add primary antibody working solutions (Anti-collagen IV antibody, catalog number ab6311; Anti-collagen VII antibody, catalog number ab6312; Anti-collagen XVII antibody, catalog number ab186415; Anti-Laminin 5 antibody, catalog number ab78286; all from abcam) and incubate overnight at 4°C. Wash three times with PBS buffer, 5 min each time. Add secondary antibody working solutions (goat anti-rabbit IgG, catalog number ab150077; goat anti-mouse IgG, catalog number ab150117; both from abcam) and incubate at room temperature for 1 h. Wash three times with PBS buffer, 5 min each time. After secondary antibody incubation, wash three times with PBS buffer, 5 min each time. Remove the PBS solution adhering to the slides, add 100 μL of Hochest working solution to each slide, and incubate at room temperature for 5 min. Wash three times with PBS buffer, 5 min each time. Wipe off the PBS solution with absorbent paper and mount the slide with a drop of anti-quenching agent. Take a fluorescence microscope image (20×) within 24 hours.

[0337] [Revised according to Rule 26, December 16, 2024] Experimental Results

[0338] [Revised according to Rule 26, December 16, 2024] 1) Results of tissue morphology experiments

[0339] [Revised according to Rule 26, December 16, 2024] The results of the epidermal viable cell layer thickness are shown in Figure 6. Compared with the blank control group (BC), the mean thickness of the epidermal viable cell layer in the negative control group (UVR irradiation) was significantly decreased ( ##p < 0.01, indicating that the stimulation conditions of this UVR radiation were effective; compared with the negative control group, the thickness of the epidermal viable cell layer treated with 100 ppm recombinant type IV collagen truncated peptide was significantly increased (**p < 0.01), with an increase rate of 63.34%, and the effect was comparable to that of the positive control, indicating that recombinant type IV collagen has a good protective effect on the skin tissue morphology against UV radiation.

[0340] [Revised according to Rule 26, December 16, 2024] 2) Dermal layer density results

[0341] [Revised according to Rule 26, December 2024] As shown in Figure 7, 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 this UVR radiation were effective; compared with the negative control group, the density of dermal fibroblasts treated with 100 ppm recombinant type IV collagen truncated peptide was significantly increased (**p < 0.01), with an increase rate of 140.82%, and the effect was comparable to that of the positive control, indicating that recombinant type IV collagen has a good protective effect on the skin tissue morphology against UV radiation.

[0342] [Revised according to Rule 26, December 2024] 3) Results of fluorescent staining experiment

[0343] [Amended according to Rule 26, December 16, 2024] The results of fluorescent staining of type IV collagen are shown in Figure 8. Compared with the blank control group (BC), the content of type IV collagen in the negative control group (UVR irradiation) was significantly decreased. ## The result (p < 0.01) indicates that the stimulation conditions of this UVR radiation were effective. Compared with the negative control group, 100 ppm of recombinant type IV collagen cleavage peptide significantly increased the content of endogenous type IV collagen in skin cells (**p < 0.01), with an increase rate of 72.09%. This indicates that recombinant type IV collagen can effectively protect against the effects of UV radiation on endogenous type IV collagen in skin cells, thereby achieving the effect of resisting photoaging.

[0344] [Amended according to Rule 26, December 16, 2024] The results of fluorescence staining of type VII collagen are shown in Figure 9. Compared with the blank control group (BC), the content of type VII collagen in the negative control group (UVR irradiation) was significantly decreased. ##The result (p < 0.01) indicates that the stimulation conditions of this UVR radiation were effective. Compared with the negative control group, 100 ppm of recombinant type IV collagen cleavage peptide significantly increased the content of endogenous type VII collagen in skin cells (**p < 0.01), with an increase rate of 80.49%, which was comparable to the positive control. This indicates that recombinant type IV collagen can effectively protect against the effects of UV radiation on endogenous type VII collagen in skin cells, thereby achieving the effect of resisting photoaging.

[0345] [Amended according to Rule 26, December 16, 2024] The results of fluorescence staining of type XVII collagen are shown in Figure 10. Compared with the blank control group (BC), the content of type XVII collagen in the negative control group (UVR irradiation) was significantly decreased. ## The result (p < 0.01) indicates that the stimulation conditions of this UVR radiation were effective. Compared with the negative control group, 100 ppm of recombinant type IV collagen significantly increased the content of endogenous type XVII collagen in skin cells (**p < 0.01), with an increase rate of 66.67%, which is comparable to the positive control. This indicates that recombinant type IV collagen can effectively protect against the effects of UV radiation on endogenous type XVII collagen in skin cells, thereby achieving the effect of resisting photoaging.

[0346] [Amended according to Rule 26, December 16, 2024] The results of laminin 5 fluorescence staining are shown in Figure 11. Compared with the blank control group (BC), the content of Laminin 5 in the negative control group (UVR irradiation) was significantly decreased. ## The result (p < 0.01) indicates that the stimulation conditions of this UVR radiation were effective. Compared with the negative control group, 100 ppm of recombinant type IV collagen cleavage peptide significantly increased the content of skin cell laminin 5 (**p < 0.01), with an increase rate of 280%, and the effect was comparable to that of the positive control group. This indicates that recombinant type IV collagen can effectively protect against the effects of UV radiation on skin cell laminin 5, thereby achieving the effect of resisting photoaging.

Claims

1. A polypeptide, characterized in that, The polypeptide contains the amino acid sequence shown in SEQ ID NO: 1 or a variant amino acid sequence obtained by substitution, deletion or insertion of one or more amino acids based on SEQ ID NO: 1; the polypeptide does not contain a full-length type IV collagen sequence.

2. The polypeptide according to claim 1, wherein it is a truncated peptide of type IV collagen; preferably, the full-length type IV collagen has the amino acid sequence shown in SEQ ID NO:

21.

3. The polypeptide according to claim 1, comprising the amino acid sequence shown in SEQ ID NO: 1 and having a length of 31-50 amino acids; preferably, having a length of 31-45 amino acids; more preferably, having a length of 31-40 amino acids.

4. The polypeptide according to claim 1, wherein 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; preferably, the total number of amino acids deleted at the N-terminus and C-terminus of SEQ ID NO: 1 does not exceed 10, 9, 8, 7, 6, 5, 4, 3, or 2.

5. The polypeptide according to claim 1, wherein the variant has activity equivalent to or better than SEQ ID NO: 1; said activity includes mitochondrial protection, skin tissue morphology protection and / or promotion of expression of endogenous anti-aging related proteins; preferably, said anti-aging related proteins include type IV collagen, type VII collagen, type XVII collagen and / or laminin 5.

6. A polynucleotide encoding a polypeptide as described in any one of claims 1-5.

7. The polynucleotide of claim 6, further comprising a sequence encoding a purification tag and / or a signal peptide sequence; Preferably, the signal peptide is a hybrid signal peptide composed of Ost1 secretory signal peptide and pro signal peptide; Preferably, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2; Preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 3 or its degenerate sequence.

8. A vector comprising the polynucleotide of claim 6 or 7; Preferably, the vector is an integrative plasmid; Preferably, the integrative plasmid further includes a methanol-inducible promoter of the AOX1 gene and / or a transcription terminator of the AOX1 gene; Preferably, the backbone sequence of the integrative plasmid includes a G418 resistance gene cassette, an ampicillin resistance gene cassette, and / or a Col E1 replicon; Preferably, 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%, or 99% identity with SEQ ID NO: 9; Preferably, 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.

9. A cell comprising the polynucleotide of claim 6 or 7 or the vector of claim 8; preferably, the cell is a eukaryotic cell; more preferably, the eukaryotic cell is a yeast host cell.

10. A composition, characterized in that, The composition comprises the polypeptide according to any one of claims 1-5; preferably, the composition is formulated into a dosage form suitable for administration by means selected from oral, topical and injection methods.

11. The use of the polypeptide according to any one of claims 1-5 or the composition according to claim 10 in the preparation of cosmetics, health products, food additives or pharmaceuticals.

12. A method for producing the polypeptide according to any one of claims 1-5, characterized in that, The process includes the following steps: fermenting and culturing the cells of claim 9, and separating and purifying the polypeptide from the culture; preferably, the cells are yeast cells.

13. The method according to claim 12, wherein, Methanol induction was performed during the yeast fermentation expression process.

14. A method for designing and screening functional protein bioactive peptides, characterized in that, Includes the following steps: 1) Recognition of receptors by functional proteins; 2) Binding site analysis: Potential binding sites on functional proteins to bind receptors were analyzed using protein docking models; 3) Peptide design: Scaffold design based on binding sites using diffusion models; 4) Sequence generation: The ProteinMPNN model is used to generate the corresponding polypeptide amino acid sequence for the polypeptide backbone designed in step 3). 5) Structural prediction: Perform three-dimensional structural prediction on the sequence generated in step 4); 6) Result screening: Based on the structural prediction results of step 5), the first candidate polypeptide group is obtained through screening; 7) Sequence alignment and candidate optimization: The first candidate polypeptide group obtained in step 6) is aligned with the known functional protein sequences to evaluate the similarity and difference between the candidate polypeptide sequences and the known functional protein sequences. The sequences are sorted according to similarity, and a target number of second candidate polypeptide groups with high similarity to the known functional protein sequences are selected. 8) Experimental verification: The second candidate polypeptide group obtained in step 7) is functionally verified. Based on the experimental data, the polypeptides are ranked according to their performance to obtain one or more target active polypeptides.

15. The method according to claim 14, wherein in step 1), potential functional protein receptors are identified by searching protein databases and literature.

16. The method according to claim 14, wherein the protein docking model in step 2) uses the CB-Dock 2 tool to perform binding site analysis on the identified receptors; preferably, the CB-Dock 2 is based on cavity detection and the AutoDock Vina algorithm to identify potential binding sites on the protein surface.

17. The method according to claim 14, wherein in step 3), 1000-3000 polypeptide backbones are designed for each binding site.

18. The method according to claim 14, wherein in step 4), 2-6 polypeptide sequences are generated for each polypeptide backbone; preferably, 4 polypeptide sequences are generated for each polypeptide backbone.

19. The method according to claim 14, wherein AlphaFold2 is used for protein three-dimensional structure prediction in step 5).

20. The method according to claim 14, wherein in step 6), the selection is based on the prediction score provided by AlphaFold2, and the selection criteria include: plddt_binder>90 and / or plddt_target>90.

21. The method according to claim 14, wherein the experiment in step 8) includes binding capacity experiment, cell experiment and / or animal experiment.

22. The method according to any one of claims 14-21, wherein the functional protein is type IV collagen; the experiments in step 8) include experiments on binding ability to integrin receptors, cell adhesion and migration experiments.

23. The method according to claim 22, wherein the active peptide obtained by screening is as described in any one of claims 1-5.

24. The application of the method according to any one of claims 14-23 in the development of cosmetic ingredients, drug development and / or basic research.

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