Recombinant small molecule collagen, expression systems, and methods for preparing this collagen.

Abstract

The invention provides a recombinant small molecule collagen, its expression system, and its preparation method, and falls within the fields of synthetic biology, genetic engineering, and biotechnology. The invention provides a recombinant small molecule collagen derived from various type III and type XVII collagen sources, and successfully expresses the recombinant small molecule collagen. The invention establishes a small molecule collagen expression system and a small molecule collagen preparation method, and includes a proprietary designed framework cell, a designed serial repeat sequence of the small molecule collagen, and a recombinant vector. The expression system or method of the invention significantly increases the yield of the small molecule collagen. The invention avoids the costs incurred by in vitro protease cleavage and the risk of foreign protein residue, and also reduces the time and cost of subsequent purification.

Description

Recombinant small molecule collagen and its expression system and preparation method Technical Field

[0001] The present invention relates to recombinant small molecule collagen and its expression system and preparation method, belonging to the fields of synthetic biology, genetic engineering and biotechnology. Background Art

[0002] Collagen has unique functional characteristics such as multiple biological functions, good biocompatibility, bioactivity and degradability. It is the most ideal source of biomaterials and can be widely used in many fields such as chemical industry, medicine, food, cosmetics, etc., with broad application prospects. Animal-derived collagen currently dominates the market. In recent years, recombinant collagen prepared by genetic engineering has begun to rise. However, whether it is animal-derived collagen or recombinant collagen, most of them are proteins with long amino acid sequences and large molecular weight. The molecular weight of animal-derived collagen is generally 100-300KDa and above, and the molecular weight of recombinant collagen is generally above 20KDa (more than 200 amino acids), with 50KDa being the most common. Such high-molecular-weight collagen is suitable for use as a biomaterial in tissue regeneration and repair, implantable medical devices, and imported medical beauty products, but it is not suitable for some application scenarios that require transdermal absorption.

[0003] Collagen with a relatively small molecular weight from animal sources is mostly small molecule collagen polypeptides, which are widely used in application scenarios that require transdermal absorption, such as small molecule collagen polypeptides from aquatic products. However, these products are mostly prepared by enzymatic hydrolysis, hydrolysis, acid-base degradation, etc. The small molecule collagen polypeptides obtained are not a small molecule protein with a clear amino acid sequence, molecular weight, and a single size, but a general term. Small molecule collagen polypeptides with molecular weights ranging from a few very small peptides to small molecule collagen polypeptides with a certain amino acid sequence length are distributed. When the type and number are almost impossible to know, the amino acid sequence is also random (unknown). The quality of small molecule collagen polypeptides prepared from different batches is also basically random, and the distribution and proportion of their amino acid sequence and molecular weight cannot be controlled. In summary, there are many studies on existing small molecule collagen products, but most of them are complexes obtained after processing (enzymatic hydrolysis, hydrolysis) of animal-derived glue. They are used in applications related to transdermal absorption, but the amino acid sequence, molecular weight, and batch differences cannot be effectively controlled.

[0004] Patent application number CN202211579848.5 discloses a small-molecule recombinant collagen peptide and its preparation method. Essentially, this method is still similar to that used for preparing animal-derived small collagen peptides. The resulting small-molecule recombinant collagen peptide is a mixture of many collagen peptides with different amino acid sequences and molecular weights (only the average molecular weight can be calculated), rather than a single recombinant small-molecule collagen protein (polypeptide) with a clear amino acid sequence and molecular weight. Other existing recombinant small-molecule collagen peptides struggle to achieve a balance between low molecular weight and biological activity. Recombinant collagens produced through synthetic biology and genetic engineering are mostly medium- and large-molecule products (some research and development results are called polypeptides, but the number of amino acids is far greater than 100, which no longer falls into the category of small molecules). Only a very small number of research results on recombinant small-molecule collagen peptides have relatively low molecular weights. As an active substance, collagen requires a certain amino acid sequence and length to support its biological activity. That is, a balance between low molecular weight and biological activity is necessary for its effectiveness. However, there is no corresponding evidence that existing small-molecule recombinant collagen peptides have good biological activity.

[0005] Similar to other small molecule proteins or polypeptides, recombinant small molecule collagen has a small number of amino acids and a small molecular weight. In genetic recombinant expression systems, especially eukaryotic expression systems (such as Pichia pastoris expression systems and mammalian cell expression systems), its expression efficiency and yield are greatly limited. There are some technical means, such as tandem expression, to increase gene copy number, but these often introduce non-essential amino acids into small molecule proteins or polypeptides. Collagen is a special protein, whose typical feature is that its amino acid sequence is a GXY triplet repeating structure. The introduction of non-essential amino acids often destroys this triplet repeating structure, affecting its functional activity.

[0006] There are few studies on existing recombinant small molecule collagen or multiple recombinant small molecule collagen polypeptides, and the products of clear recombinant small molecule collagen are basically blank, and the problem of expression level involves even less. There are studies using the method of fusion leader peptide to improve the expression level of small molecule protein, such as the patent with publication number CN108148114B, but the small molecule protein that needs to be expressed can be removed by enzymatic cleavage with protease in vitro (cell), and exogenous protein is artificially introduced into the expression product, which increases the step and cost of subsequent purification process and also brings the risk of exogenous protein residue. In addition, there are some studies using the method of serial expression of small molecule protein to improve expression efficiency to achieve the purpose of increasing output, such as CN110305890A, which uses 5 kinds of antimicrobial peptides to express and reorganize in series, but uses formic acid to cut the polypeptide of series connection, and the cleavage site can only be between two amino acid residues of DP, so the design space is limited, and after cutting, DP cannot be effectively removed and becomes non-self amino acid, which also belongs to artificial introduction of exogenous protein. These schemes are also not seen to be used for the expression and production of recombinant small molecule collagen.

[0007] Therefore, the expression of recombinant small molecule collagen in this technical field needs to achieve a balance in many aspects, such as small molecular weight, biological activity, scalable mass production, high expression level, and a single and clear amino sequence and molecular weight rather than a mixture. An effective solution is urgently needed.

[0008] Summary of the Invention

[0009] The purpose of the present invention is to overcome some technical problems existing in the prior art and provide a recombinant small molecule collagen, an expression system and a preparation method thereof.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] The present invention first provides a recombinant small molecule collagen, which includes the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.63 or SEQ ID NO.67, or an amino acid sequence that has more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identity with SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.63 or SEQ ID NO.67, and maintains the biological activity of the collagen.

[0012] Furthermore, the carboxyl terminus of the recombinant small molecule collagen has multiple His residues, which can form a 6×His Tag tag.

[0013] Furthermore, the recombinant small molecule collagen includes the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8, or an amino acid sequence with more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identity with SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8, and maintains the biological activity of collagen.

[0014] The present invention also provides a nucleic acid encoding the recombinant small molecule collagen.

[0015] Furthermore, the nucleic acid includes the nucleotide sequence shown in SEQ ID NO. 9-16, or a degenerate sequence thereof.

[0016] The present invention also provides a recombinant vector comprising the nucleic acid, wherein the vector comprises pPICZαB, pFLDα, and pPIC9K, and the connection site is between the Kex2 restriction enzyme cleavage site sequence and the NotI restriction enzyme cleavage site.

[0017] The present invention also provides a recombinant engineered bacterium comprising the nucleic acid, or comprising the recombinant vector, or expressing the recombinant small molecule collagen.

[0018] Furthermore, the host bacteria of the engineered bacteria include Pichia pastoris, Saccharomyces cerevisiae, Hansenula, etc., preferably Pichia pastoris.

[0019] According to an embodiment of the present invention, the engineered bacteria are deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit numbers being CGMCC No. 25811, CGMCC No. 25823, CGMCC No. 25812, CGMCC No. 25824, CGMCC No. 25825, CGMCC No. 25813, CGMCC No. 25826, and CGMCC No. 25814.

[0020] The present invention also provides a tandemly repeated expression sequence of a recombinant small molecule collagen protein, wherein the tandemly repeated expression sequence is based on the recombinant small molecule collagen protein, or an artificially designed collagen protein having a typical GXY triplet structure, or a protein composed of two or more regions of a human collagen protein sequence and is repeated in tandem; between each two adjacent recombinant small molecule collagen proteins in the tandemly repeated expression sequence, there are sites for recognition and cleavage by the Kex2 enzyme and the CPB enzyme, and there may also be sites for recognition and cleavage by the Ste13 enzyme.

[0021] The tandem repeat expression sequence includes the sequences shown in SEQ ID NO.30, SEQ ID NO.33, SEQ ID NO.36, SEQ ID NO.39, SEQ ID NO.42, SEQ ID NO.45, SEQ ID NO.48, SEQ ID NO.51, SEQ ID NO.54, SEQ ID NO.57, SEQ ID NO.60, SEQ ID NO.64, and SEQ ID NO.68, or amino acid sequences having more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identity thereto.

[0022] According to an embodiment of the present invention, the site recognized and cleaved by the Kex2 enzyme includes a KR or RR dibasic amino acid residue, followed by EA, EAEA or other amino acid residues that are helpful for the recognition and cleavage of the Kex2 enzyme or Ste 13 enzyme.

[0023] According to an embodiment of the present invention, the sites recognized and cleaved by the CPB enzyme include basic amino acid residues at the carboxyl terminus of the protein, including K and R.

[0024] According to an embodiment of the present invention, the amino acid sequence of the tandemly repeated small molecule collagen monomer (or fragment) can be any sequence, and the specific order of its amino acid residues, sequence length, and the number of tandem repetitions of the monomer (or fragment) are not restricted, but it must have a typical GXY triplet structure.

[0025] The present invention also provides a nucleic acid encoding the tandemly repeated expression sequence. Furthermore, the nucleic acid comprises the sequence shown in SEQ ID NO. 71-81, or a degenerate sequence thereof.

[0026] The present invention also provides a recombinant vector comprising a nucleic acid encoding the tandemly repeated expression sequence. According to an embodiment of the present invention, the vector includes but is not limited to expression vectors such as pPICZαB, pFLDα, and pPIC9K.

[0027] The present invention also provides an engineered bacterium comprising the nucleic acid or recombinant vector.

[0028] According to an embodiment of the present invention, the host bacteria of the engineered bacteria include Pichia pastoris, Saccharomyces cerevisiae, Hansenula, etc., preferably Pichia pastoris. According to an embodiment of the present invention, the engineered bacteria are deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with the deposit numbers being: CGMCC No. 25819, CGMCC No. 25821, CGMCC No. 25827, CGMCC No. 25829, CGMCC No. 25828, CGMCC No. 25820, and CGMCC No. 25822.

[0029] The present invention also provides a method for obtaining recombinant small molecule collagen, the method comprising:

[0030] Constructing the tandem repeat expression sequence of the recombinant small molecule collagen of the present invention;

[0031] Constructing a localization fusion functional protein, connecting the localization fusion functional protein to a vector and transforming the host bacteria to obtain chassis cells or chassis engineered bacteria;

[0032] The tandem repeat expression sequence of the recombinant small molecule collagen is connected to an expression vector and then transferred into chassis cells or chassis engineered bacteria to obtain recombinant engineered bacteria containing or expressing the recombinant small molecule collagen. The expression is induced by fermentation to obtain the recombinant small molecule collagen.

[0033] According to an embodiment of the present invention, the localization fusion functional protein includes a CPB enzyme and a functional region having intracellular membrane localization or conversion and transport functions between organelles.

[0034] Furthermore, the localization fusion functional protein also includes a linker sequence, which is used to connect the CPB enzyme with the functional region sequence that has intracellular membrane localization or conversion and transport between organelles when fused and expressed. Preferably, the linker sequence is a linker sequence, such as that shown in SEQ ID NO. 21: GGSGSGSGGS.

[0035] According to an embodiment of the present invention, preferably, the CPB enzyme is derived from human or rat; the CPB enzyme sequence is shown in SEQ ID NO.17-18.

[0036] The functional region sequence having the function of intracellular membrane localization or conversion and transport between organelles is derived from Kex2 enzyme of Saccharomyces cerevisiae or Pichia pastoris, or other protein functional regions with similar functions such as Ste13 protease.

[0037] Furthermore, the sequences of the functional regions having intracellular membrane localization or conversion and transport functions between organelles are shown in SEQ ID NOs. 19-20.

[0038] According to an embodiment of the present invention, there are recognition and cleavage sites for Kex2 enzyme and CPB enzyme between each two adjacent recombinant small molecule collagen proteins in the tandemly repeated expression sequence, and may also include recognition and cleavage sites for Ste13 enzyme; the small molecule collagen protein is preferably the recombinant small molecule collagen protein described in the present invention.

[0039] According to an embodiment of the present invention, the site recognized and cleaved by the Kex2 enzyme includes a KR or RR dibasic amino acid residue, followed by EA, EAEA or other amino acid residues that are helpful for the recognition and cleavage of the Kex2 enzyme or Ste 13 enzyme.

[0040] According to an embodiment of the present invention, the sites recognized and cleaved by the CPB enzyme include basic amino acid residues at the carboxyl terminus of the protein, including K and R.

[0041] Furthermore, the recombinant small molecule collagen has a typical GXY triplet structure; preferably, the small molecule collagen is the recombinant small molecule collagen described in the present invention, or an artificially designed collagen with a typical GXY triplet structure, or a collagen composed of two or more regions of a human collagen sequence.

[0042] According to an embodiment of the present invention, the vector includes but is not limited to expression vectors such as pPICZαB, pFLDα, and pPIC9K.

[0043] According to an embodiment of the present invention, the recombinant engineered bacteria containing or expressing recombinant small molecule collagen are deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit numbers being: CGMCC No.25819, CGMCCNo.25821, CGMCC No.25827, CGMCC No.25828, CGMCC No.25829, CGMCC No.25820, and CGMCC No.25822.

[0044] According to an embodiment of the present invention, the recombinant small molecule collagen expressed by the above method includes the amino acid sequence shown in SEQ ID NO.32, SEQ ID NO.35, SEQ ID NO.38, SEQ ID NO.41, SEQ ID NO.44, SEQ ID NO.47, SEQ ID NO.50, SEQ ID NO.53, SEQ ID NO.56, SEQ ID NO.59, SEQ ID NO.62, SEQ ID NO.66, or SEQ ID NO.70, or the amino acid sequence shown in SEQ ID NO.32, SEQ ID NO.35, SEQ ID NO.38, SEQ ID NO.41, SEQ ID NO.44, SEQ ID NO.47, SEQ ID NO.50, SEQ ID NO.53, SEQ ID NO.56, SEQ ID NO.59, SEQ ID NO.62, SEQ ID NO.66, or SEQ ID NO.70. NO.70 has an amino acid sequence with an identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0045] The present invention also provides a localized fusion functional protein, which is used for cleaving and removing the C-terminal basic amino acid residues (K, R) of multiple tandemly repeated recombinant small molecule collagens after they are cleaved into monomers by Kex2 enzyme.

[0046] The localization fusion functional protein comprises a CPB enzyme mature peptide sequence and a functional region sequence having intracellular membrane localization or conversion and transport functions between organelles.

[0047] Furthermore, the localization fusion functional protein also includes a connecting sequence, which is used for connecting the CPB enzyme with the functional region sequence having intracellular membrane localization or conversion and transport functions between organelles when fused and expressed.

[0048] According to an embodiment of the present invention, the CPB enzyme is derived from humans or rats; further, the CPB enzyme is preferably a sequence shown in SEQ ID NO. 17-18.

[0049] According to an embodiment of the present invention, the functional region sequence having intracellular membrane localization or conversion and transport between organelles is derived from Kex2 enzyme of Saccharomyces cerevisiae or Pichia pastoris, or other protein functional regions with similar functions such as Ste13 protease.

[0050] Furthermore, the CPB enzyme sequence is shown in SEQ ID NOs. 17-18; the functional region sequence having intracellular membrane localization or conversion and transport between organelles is shown in SEQ ID NOs. 19-20.

[0051] According to an embodiment of the present invention, the connecting sequence is a linker sequence, as shown in SEQ ID NO. 21: GGSGSGSGGS.

[0052] According to an embodiment of the present invention, the positioning fusion functional protein includes an amino acid sequence as shown in SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26 or SEQ ID NO.28, or an amino acid sequence that is more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identical to SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26 or SEQ ID NO.28.

[0053] The present invention also provides a nucleic acid encoding the localization fusion functional protein of the present invention.

[0054] According to an embodiment of the present invention, the nucleic acid encoding the localized fusion functional protein of the present invention includes the nucleotide sequence shown in SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27 or SEQ ID NO.29, or a degenerate sequence thereof.

[0055] The present invention also provides a recombinant vector comprising a nucleic acid encoding the localized fusion functional protein of the present invention.

[0056] According to an embodiment of the present invention, the vector includes but is not limited to expression vectors such as pPICZαB, pFLDα, and pPIC9K, and the connection position is between the Kex2 restriction site sequence and the termination codon "TGA" on the vector.

[0057] The present invention also provides a chassis cell or chassis engineered bacteria, which contains the nucleic acid encoding the localization fusion functional protein of the present invention, or a recombinant vector containing the nucleic acid encoding the localization fusion functional protein of the present invention, or expresses the localization fusion functional protein.

[0058] According to an embodiment of the present invention, the host bacteria of the engineered bacteria include Pichia pastoris, Saccharomyces cerevisiae, Hansenula, etc., preferably Pichia pastoris; the constructed chassis cells or engineered chassis bacteria are deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with the deposit numbers being: CGMCC No. 25815, CGMCC No. 25817, CGMCC No. 25816, and CGMCC No. 25818.

[0059] The present invention also provides an expression system for recombinant small molecule collagen, which includes the positioning fusion functional protein, the chassis cells or chassis engineering bacteria, and the tandem repeat expression sequence of the recombinant small molecule collagen.

[0060] The present invention also provides the use of the localized fusion functional protein, the chassis cells or chassis engineering bacteria, and the tandem repeat expression sequence of the recombinant small molecule collagen in expressing the recombinant small molecule collagen.

[0061] The recombinant small molecule collagen of the present invention or the small molecule collagen expressed by the above method for obtaining small molecule collagen has cell adhesion activity, cell proliferation promoting activity, and good skin permeability.

[0062] The present invention also provides uses of the recombinant small molecule collagen, or the nucleic acid, or the recombinant vector, or the host cell or recombinant engineered bacteria, or the composition, or the product in the preparation of drugs, medical devices, biomaterials, tissue engineering products, cosmetics, or health products.

[0063] Beneficial effects of the present invention:

[0064] (1) The present invention first provides a variety of recombinant small molecule collagens derived from type III and type XVII collagen, and expresses a variety of recombinant small molecule collagens derived from type III and type XVII collagen. Although the amino acid sequence lengths and molecular weights are different, they can all be expressed as small molecule collagens with a single band and a clear amino acid sequence. At the same time, they all have good transdermal absorption and biological activity, and achieve a balance between small molecules and transdermal absorption and biological activity. Further, large-scale mass production has been achieved in a 500L fermentation tank, making it possible to apply recombinant small molecule collagens on a large scale. This solves the problem of the lack of existing recombinant small molecule collagen products, especially recombinant small molecule collagens derived from type III and type XVII collagen.

[0065] (2) The present invention establishes an expression system for recombinant small molecule collagen and a method for preparing small molecule collagen, including the construction of exclusive chassis cells and a method for designing collagen tandem repeat sequences. The technical solution of the present invention greatly increases the yield of small molecule collagen expression; the technical solution of the present invention does not introduce exogenous proteins and does not use any in vitro enzyme cleavage methods. During the process of intracellular secretion of small molecule collagen, the residual amino acid residues at the enzyme cleavage site during the repeated tandem design will be removed, and a recombinant small molecule collagen with no non-collagen sequence or 100% homology to the corresponding region of natural collagen will be obtained; the entire expression system avoids the cost and risk of exogenous protein residues caused by in vitro protease cleavage, and can also shorten the time and cost of the subsequent purification process.

[0066] The small molecule collagen obtained by the present invention was verified by N-terminal, C-terminal and full sequence sequencing, proving the effectiveness of the entire expression system, especially the chassis cell engineering bacteria as host bacteria to express recombinant small molecule collagen; further fermentation and purification verification with the possibility of large-scale industrial production was carried out in a 500L fermenter. The results showed that the expression system constructed by the repeated tandem expression strategy can increase the yield of recombinant small molecule collagen by 4-5 times.

[0067] (3) The cell adhesion activity, cell proliferation promoting activity and transdermal absorption of the recombinant small molecule of the present invention were tested. The results showed that in addition to having good transdermal absorption, the recombinant small molecule collagen also has cell adhesion activity that is not inferior to that of natural collagen and homologous high molecular weight recombinant collagen, and has cell proliferation promoting activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 shows the results of Tricine-PAGE detection of 8 recombinant small molecule collagens 3A5D1NT, 3A5D1, 3A5D2NT, 3A5D2, 17S1NNT, 17S1N, 17S3NT, and 17S3 after purification.

[0069] FIG2 shows the deconvoluted molecular weight results of the recombinant small molecule collagens 3A5D1NT and 3A5D1 expressed by the present invention obtained by LC-MS analysis.

[0070] FIG3 shows the deconvoluted molecular weight of the recombinant small molecule collagen 3A5D2 expressed by the present invention obtained by LC-MS analysis.

[0071] FIG4 shows the deconvoluted molecular weight results of the recombinant small molecule collagens 17S1NNT and 17S1N expressed by the present invention obtained by LC-MS analysis.

[0072] FIG5 shows the deconvoluted molecular weight results of the recombinant small molecule collagens 17S3NT and 17S3 expressed by the present invention obtained by LC-MS analysis.

[0073] FIG6 shows the results of Western Blot detection of the carboxyl-terminal 6×His tags of 3A5D1, 3A5D2, 17S1N, and 17S3 using an anti-6×His Tag antibody; in the figure, + indicates a positive control, and - indicates a negative control.

[0074] FIG7 shows the results of comparison of peptide segments of the recombinant small molecule collagen 3A5D1 and 3A5D1NT freeze-dried products digested with trypsin and detected by Nano-HPLC-MS / MS mass spectrometry with the original sequence in Example 1 of the present invention.

[0075] FIG8 is a result of comparison between the peptide segments of the recombinant small molecule collagen 3A5D2 and 3A5D2NT freeze-dried products digested with trypsin and the original sequence after Nano-HPLC-MS / MS mass spectrometry detection in Example 1 of the present invention.

[0076] FIG9 shows the results of comparison of peptide segments of the recombinant small molecule collagen 17S1N and 17S1NNT freeze-dried products digested with trypsin and detected by Nano-HPLC-MS / MS mass spectrometry with the original sequence in Example 1 of the present invention.

[0077] FIG10 is a result of comparison between the peptide segments of the recombinant small molecule collagen 17S3 and 17S3NT freeze-dried products digested with trypsin and the original sequence after Nano-HPLC-MS / MS mass spectrometry detection in Example 1 of the present invention.

[0078] FIG11 is a map of the recombinant expression vector pPICZαB-RCPB-SCKEX2 of the present invention.

[0079] FIG12 is a map of the recombinant expression vector pPICZαB-HCPB-SCKEX2 of the present invention.

[0080] FIG13 is a map of the recombinant expression vector pPICZαB-RCPB-PPKEX2 of the present invention.

[0081] FIG14 is a map of the recombinant expression vector pPICZαB-HCPB-PPKEX2 of the present invention.

[0082] Figure 15 shows the expression detection results of CPB localization fusion functional proteins HCPB-PPKEX2, HCPB-SCKEX2, RCPB-PPKEX2, and RCPB-SCKEX2 in intracellular lysates and culture supernatants;

[0083] Lanes in the left figure: 1: HCPB-SCKEX2 bacterial lysate; 2: HCPB-SCKEX2 bacterial culture supernatant; 3, 6: HCPB-PPKEX2 culture supernatant; 4, 5: HCPB-PPKEX2 bacterial lysate; (-): negative control; (+): positive control, recombinant human CPB enzyme; / : well without sample.

[0084] Lanes in the right figure, (-): negative control; (+): positive control, recombinant rat CPB enzyme; / : well without sample; 7: RCPB-SCKEX2 bacterial culture supernatant; 8: RCPB-SCKEX2 bacterial lysate; 9, 10, 12, 13: RCPB-PPKEX2 culture supernatant; 11, 14: RCPB-PPKEX2 bacterial lysate.

[0085] Figure 16 shows the SDS-PAGE analysis results of the supernatants of 3A5D29N-9 and 17S28-8 expressed in HCPB-PPKEX2 and RCPB-PPKEX2 chassis cell engineering strains, respectively;

[0086] The lanes in the figure are: 1: supernatant of 3A5D29N-9 expressed in HCPB-PPKEX2 chassis cell engineering strain; 2: supernatant of 3A5D29N-9 expressed in RCPB-PPKEX2 chassis cell engineering strain; 3: supernatant of 17S28-8 expressed in HCPB-PPKEX2 chassis cell engineering strain; 4: supernatant of 17S28-8 expressed in RCPB-PPKEX2 chassis cell engineering strain.

[0087] Figure 17 shows the SDS-PAGE detection results of the supernatants of 17S1N6-6, 17S1N7-7 and 17S1NK-7 expressed in HCPB-PPKEX2 and RCPB-PPKEX2 chassis cell engineering strains, respectively;

[0088] The lanes in the figure are, 1: supernatant of 17S1N6-6 expressed in the HCPB-PPKEX2 chassis cell engineering strain; 2: supernatant of 17S1N6-6 expressed in the RCPB-PPKEX2 chassis cell engineering strain; 3: supernatant of 17S1N7-7 expressed in the HCPB-PPKEX2 chassis cell engineering strain; 4: supernatant of 17S1N7-7 expressed in the RCPB-PPKEX2 chassis cell engineering strain; 5: supernatant of 17S1NK-7 expressed in the HCPB-PPKEX2 chassis cell engineering strain; 6: supernatant of 17S1NK-7 expressed in the RCPB-PPKEX2 chassis cell engineering strain; (-): negative control, supernatant of expression of the pPIC9K empty vector transformed into the HCPB-PPKEX2 chassis cell engineering strain.

[0089] Figure 18 shows the SDS-PAGE analysis results of the supernatants of 3A5D15D-5, 3A5D15E-5, 3A5D15G-6 and 3A5D15EKR-6 expressed in the RCPB-PPKEX2 chassis cell engineering strain, and 3A5D15R-6 expressed in the HCPB-RPKEX2 chassis cell engineering strain;

[0090] The lanes in the figure are, 1: supernatant of 3A5D15D-5 expressed in RCPB-PPKEX2 chassis cell engineering strain; 2: supernatant of 3A5D15E-5 expressed in RCPB-PPKEX2 chassis cell engineering strain; 3: supernatant of 3A5D15G-6 expressed in RCPB-PPKEX2 chassis cell engineering strain; 4: supernatant of 3A5D15EKR-6 expressed in RCPB-PPKEX2 chassis cell engineering strain; 5: supernatant of 3A5D15KR-6 expressed in RCPB-PPKEX2 chassis cell engineering strain; 6: supernatant of 3A5D15R-6 expressed in HCPB-RPKEX2 chassis cell engineering strain; (-): negative control, supernatant of expression of pPIC9K empty vector transformed into RCPB-RPKEX2 chassis cell engineering strain.

[0091] Figure 19 shows the SDS-PAGE analysis results of the supernatants of 3A5D15D-5, 3A5D15E-5, 3A5D15G-6, A5D15EKR-6, 3A5D15KR-6, and 3A5D15R-6 expressed in the HCPB-PPKEX2 chassis cell engineering strain;

[0092] The lanes in the figure are, 1: supernatant of 3A5D15D-5 expressed in the HCPB-PPKEX2 chassis cell engineering strain; 2: supernatant of 3A5D15E-5 expressed in the HCPB-PPKEX2 chassis cell engineering strain; 3: supernatant of 3A5D15G-6 expressed in the HCPB-PPKEX2 chassis cell engineering strain; 4: supernatant of 3A5D15EKR-6 expressed in the HCPB-PPKEX2 chassis cell engineering strain; 5: supernatant of 3A5D15KR-6 expressed in the HCPB-PPKEX2 chassis cell engineering strain; 6: supernatant of 3A5D15R-6 expressed in the HCPB-PPKEX2 chassis cell engineering strain.

[0093] FIG20 shows the results of alignment of peptides of the lyophilized products of 3A5D29N and 33A5D15D with the original sequences after trypsin digestion and detection by Nano-HPLC-MS / MS mass spectrometry.

[0094] Figure 21 shows the results of alignment of peptides from the lyophilized products of 17S28 and 17S1N6 with the original sequences after trypsin digestion and detection by Nano-HPLC-MS / MS mass spectrometry.

[0095] FIG22 shows the deconvoluted molecular weight results of 3A5D29N and 17S28 obtained by LC-MS analysis.

[0096] Figure 23 is a secondary mass spectrum of the C-terminal peptide of the recombinant small molecule collagen 3A5D29N expressed in the HCPB-PPKEX2 and RCPB-PPKEX2 chassis cell engineering strains.

[0097] Figure 24 is a secondary mass spectrum of the C-terminal peptide of the recombinant small molecule collagen 17S28 expressed in the HCPB-PPKEX2 and RCPB-PPKEX2 chassis cell engineering strains.

[0098] FIG25 is the SDS-PAGE detection results of the fermentation supernatant of the engineered strains expressing 3A5D2NT and 3A5D29N after induction for a certain period of time.

[0099] The lanes in the figure are: Lane 1: fermentation supernatant of the engineered strain (CGMCC No. 25812) expressing 3A5D2NT at 48 h of induction; Lane 2: fermentation supernatant of the engineered strain (CGMCC No. 25819) expressing 3A5D29N at 43 h of induction.

[0100] Figure 26 is a comparison of the cell adhesion activities of recombinant small molecule collagens 3A5D29N, 3A5D2NT, 3A5D1NT, 17S1NNT, 17S3NT, 17S28, recombinant type III collagen, recombinant type XVII collagen, and natural human collagen.

[0101] FIG27 is the verification result of the cell proliferation promoting effect of recombinant small molecule collagen 3A5D29N and 17S28.

[0102] Figure 28 shows the average fluorescence IOD values ​​of 1 mg / mL recombinant small molecule collagen 3A5D29N, recombinant type III collagen, hydrolyzed hyaluronic acid, and fish skin collagen detected in the full-thickness skin model tissue 1 hour after application, where * indicates the significance compared with 1 mg / mL recombinant small molecule collagen 3A5D29N (* indicates a significant difference P<0.05, ** indicates an extremely significant difference P<0.01), and # indicates the significance compared with recombinant type III collagen (# indicates a significant difference P<0.05, ## indicates an extremely significant difference P<0.01).

[0103] Figure 29 shows the diffusion percentage (i.e., cumulative permeability) of samples infiltrated into culture medium after smearing with different concentrations of recombinant small molecule collagen 3A5D29N and 1 mg / mL hydrolyzed hyaluronic acid for 12 hours, where 2# represents recombinant small molecule collagen 3A5D29N and 4# represents hydrolyzed hyaluronic acid. DETAILED DESCRIPTION

[0104] In order to enable those skilled in the art to better understand the technical solutions of the present invention, preferred embodiments of the present invention are described in detail below. However, the following embodiments do not limit the scope of protection of the present invention.

[0105] In the embodiments of the present invention, those that are not described in detail are all completed using conventional experimental methods. Those processes involved in the embodiments that are not described in detail are all understandable and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore are not described in detail.

[0106] Example 1: Single sequence recombinant expression of recombinant small molecule collagen

[0107] (1) Amino acid sequence design of single sequence recombinant expression strategy for recombinant small molecule collagen

[0108] The amino acid sequence of positions 990-1085 of the full-length human type III collagen (including N propeptide, mature peptide chain, and C propeptide) was selected (referring to the protein sequence with ID P02461 in the Uniprot database, https: / / www.uniprot.org / uniprotkb / P02461). The recombinant small molecule collagen protein obtained by expressing this sequence was named 3A5D1NT. It has a total of 96 amino acids and a theoretical molecular weight of 8596.08 Da. Its sequence is shown in SEQ ID NO.1:

[0109] Five histidines (H) were added to the carboxyl terminus of the 3A5D1NT sequence. The recombinant small molecule collagen protein obtained by expressing this sequence was named 3A5D1. It has a total of 101 amino acids and a theoretical molecular weight of 9281.78 Da. Its sequence is shown in SEQ ID NO. 2:

[0110] The full-length human type III collagen (including N propeptide, mature peptide chain, and C propeptide) sequence (referenced to the protein sequence with ID P02461 in the Uniprot database, https: / / www.uniprot.org / uniprotkb / P02461) from amino acids 1036 to 1085 was selected. The recombinant small molecule collagen protein obtained by expressing this sequence was named 3A5D2NT. It has a total of 50 amino acids and a theoretical molecular weight of 4532.83 Da. Its sequence is shown in SEQ ID NO. 3:

[0111] Five histidines (H) were added to the carboxyl terminus of the 3A5D2NT sequence. The recombinant small molecule collagen protein expressed from this sequence was named 3A5D2. It has a total of 55 amino acids and a theoretical molecular weight of 5218.54 Da. Its sequence is shown in SEQ ID NO. 4:

[0112] The full-length human collagen XVII sequence (protein with ID Q9UMD9-1 in the Uniprot database, https: / / www.uniprot.org / uniprot / Q9UMD9) was selected from amino acids 636-718. The recombinant small molecule collagen protein obtained by expressing this sequence was named 17S1NNT. It has a total of 82 amino acids and a theoretical molecular weight of 7483.28 Da. Its sequence is shown in SEQ ID NO. 5:

[0113] Six histidines (H) were added to the carboxyl terminus of the 17S1NNT sequence. The recombinant small molecule collagen protein expressed from this sequence was named 17S1N. It has a total of 88 amino acids and a theoretical molecular weight of 8306.13 Da. Its sequence is shown in SEQ ID NO.6:

[0114] The full-length human collagen XVII sequence (protein with ID Q9UMD9-1 in the Uniprot database, https: / / www.uniprot.org / uniprot / Q9UMD9) was selected from amino acids 659-717. The recombinant small molecule collagen protein obtained by expressing this sequence was named 17S3NT. It has a total of 59 amino acids and a theoretical molecular weight of 5406.08 Da. Its sequence is shown in SEQ ID NO. 7:

[0115] Six histidines (H) were added to the carboxyl terminus of the 17S3NT sequence. The recombinant small molecule collagen protein expressed from this sequence was named 17S3. It has a total of 65 amino acids and a theoretical molecular weight of 6228.93 Da. Its sequence is shown in SEQ ID NO.8:

[0116] (2) Construction of recombinant expression vectors and engineering strains for the single sequence recombinant expression strategy of recombinant small molecule collagen

[0117] The DNA sequence encoding 3A5D1NT was codon-optimized using Pichia pastoris as the host. The optimized sequence is shown in SEQ ID NO.9:

[0118] The DNA sequence encoding 3A5D1 was codon-optimized using Pichia pastoris as the host. The optimized sequence is shown in SEQ ID NO.10:

[0119] The DNA sequence encoding 3A5D2NT was codon-optimized using Pichia pastoris as the host. The optimized sequence is shown in SEQ ID NO.11:

[0120] The DNA sequence encoding 3A5D2 was codon-optimized using Pichia pastoris as the host. The optimized sequence is shown in SEQ ID NO.12:

[0121] The DNA sequence encoding 17S1NNT was codon-optimized using Pichia pastoris as the host. The optimized sequence is shown in SEQ ID NO. 13:

[0122] The DNA sequence encoding 17S1N was codon-optimized using Pichia pastoris as the host. The optimized sequence is shown in SEQ ID NO. 14:

[0123] The DNA sequence encoding 17S3NT was codon-optimized using Pichia pastoris as the host. The optimized sequence is shown in SEQ ID NO. 15:

[0124] The DNA sequence encoding 17S3 was codon-optimized using Pichia pastoris as the host. The optimized sequence is shown in SEQ ID NO. 16:

[0125] DNA sequences SEQ ID NO. 9 to SEQ ID NO. 16 were synthesized by Nanjing GenScript Biotechnology Co., Ltd. The synthesized genes were cloned into the pPIC9K expression vector (Thermo Fisher Scientific (China) Co., Ltd.) between the Kex2 restriction site sequence (the DNA sequence at the end of the Kex2 restriction site is AAAGAAGAAGGGGTATCTCTCGAGAAAAGA) and the NotI restriction site within the α-factor secretion signal (secretion signal peptide) sequence. Recombinant expression vectors pPIC9K-3A5D1NT, pPIC9K-3A5D1, pPIC9K-3A5D2NT, pPIC9K-3A5D2, pPIC9K-17S1NNT, pPIC9K-17S1N, pPIC9K-17S3NT, and pPIC9K-17S3 were obtained. Theoretically, other expression vectors that can be used in Pichia pastoris, such as pPICZαB and pFLDα, also have similar effects to pPIC9K, and the corresponding recombinant expression vectors are within the scope of protection of the present invention.

[0126] 10 μg of the above-mentioned recombinant expression vector plasmids (pPIC9K-3A5D1NT, pPIC9K-3A5D1, pPIC9K-3A5D2NT, pPIC9K-3A5D2, pPIC9K-17S1NNT, pPIC9K-17S1N, pPIC9K-17S3NT, pPIC9K-17S3) were respectively digested with SacⅠ (purchased from Dalian TaKaRa Company, the specific operation was carried out according to the kit instructions) at 37°C overnight to linearize them, and then the linearized plasmid was recovered using a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to control the volume to about 10 μL.

[0127] The linearized plasmid was electroporated into the competent cells of the empty host strain Pichia pastoris GS115 (purchased from the China Industrial Microbiological Culture Collection Center). The electroporated bacterial solution was spread on MD plates, with 100 μL to 200 μL spread on one plate. The plate was allowed to stand at room temperature for 10 minutes and then inverted and cultured at 30°C for 2-5 days until a single colony (positive transformant) appeared.

[0128] Add 2 mL of sterile double-distilled water to the surface of the MD plate, and then gently scrape off the His on the surface of the plate with a sterile triangular spreader. +Transformants were isolated and transferred to a 50 mL centrifuge tube. The bacterial suspension was diluted with sterile double-distilled water, and 105 cells were plated onto a YPD plate containing 0.5 mg / mL G418. The plate was inverted and incubated at 30°C for 3-4 days until single colonies appeared. Colonies were picked from the YPD plate and transferred to a sterile 96-well plate (200 μL YPD / well), mixed, and incubated at 30°C for 48 hours. The bacterial suspension in each well was mixed and 10 μL was transferred to a new sterile 96-well plate. The plate was incubated at 30°C for 24 hours, and this process was repeated once. After 24 hours, 1 μL of the solution was taken from the third 96-well plate and spotted onto YPD plates containing 1.0 mg / mL and 4 mg / mL G418, respectively. The culture was continued at 30°C for 96-120 hours. If the Pichia pastoris transformant can grow on a plate containing a high concentration of G418, it means that the transformant can express the exogenous gene with high efficiency. After this step of screening, a recombinant yeast engineered strain with high expression efficiency can be obtained.

[0129] The eight engineered strains were deposited in the General Microbiology Center of the China Culture Collection Administration. The corresponding deposit numbers are:

[0130] The strain expressing the recombinant small molecule collagen 3A5D1NT has the deposit number: CGMCC No.25823;

[0131] The strain expressing the recombinant small molecule collagen 3A5D1 has the deposit number: CGMCC No.25811;

[0132] The strain expressing the recombinant small molecule collagen 3A5D2NT has the deposit number: CGMCC No.25824;

[0133] The strain expressing the recombinant small molecule collagen 3A5D2 has the deposit number: CGMCC No.25812;

[0134] The strain expressing the recombinant small molecule collagen 17S1NNT has the deposit number: CGMCC No.25825;

[0135] The strain expressing the recombinant small molecule collagen 17S1N has the deposit number: CGMCC No.25813;

[0136] The strain expressing the recombinant small molecule collagen 17S3NT has the deposit number: CGMCC No.25826;

[0137] The strain expressing recombinant small molecule collagen 17S3 has the deposit number: CGMCC No.25814;

[0138] The deposit address of the above eight engineered bacteria is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is: September 26, 2022. The taxonomic name is: Pichia pastoris Komagataella phaffii.

[0139] (3) Expression, purification and identification of recombinant small molecule collagen single sequence recombinant expression strategy

[0140] The eight highly efficient recombinant yeast engineered strains obtained in step (2) were used to perform high-density fermentation and expression of recombinant small molecule collagen using a fed-batch culture method to obtain a fermentation broth containing recombinant collagen, which was then purified to obtain a high-purity recombinant small molecule collagen freeze-dried sponge. The specific steps are as follows:

[0141] The eight highly expressed recombinant yeast strains obtained in step (2) were selected and inoculated into 1 L shake flasks containing seed medium YPG, cultured at 220 rpm and 30 ° C for 18-20 h until OD 600 = 2-10; Inoculate 200 mL of seed solution into a 5-L fermenter (purchased from Shanghai Baoxing Bio-Equipment Engineering Co., Ltd.) and fill with 2 L of fermentation medium. Induce the culture with a mixed carbon source of methanol and glycerol (methanol: 50% glycerol = 7:3). After 40-60 hours of induction, release the tank if the protein concentration measured by UV does not increase significantly or decreases. The formula for UV protein concentration is: C (mg / mL) = 0.144 * (A215 - A225).

[0142] The fermentation broth was collected and centrifuged at 2000g for 30min at 4°C to separate the bacteria and the fermentation supernatant. The fermentation supernatant was purified by cation exchange chromatography (the chromatography filler was UniGel-80sp produced by Suzhou Nano Micro, loaded on a GCC-50-400 chromatography column produced by Lisui Technology, using a GE AKTA Pure protein separation chromatography purification system), followed by ultrafiltration (using a 1KDa organic membrane filtration system from Shandong Bona Biotechnology Group Co., Ltd., model BNUF402-2-A), desalting, concentration, and subsequent freeze-drying to collect the recombinant small molecule collagen freeze-dried sponge.

[0143] The purified lyophilized sponge was dissolved in ultrapure water, 2× small molecule protein loading buffer was added, and the sample was heated in a 100°C metal bath for 10 minutes. Tricine-PAGE analysis was then performed (the complete set of reagents required for Tricine-PAGE electrophoresis for small molecule proteins was purchased from Shanghai Wansheng Haotian Biotechnology Co., Ltd., and the electrophoresis method was performed according to the instructions). The test results are shown in Figure 1. As can be seen in the figure, the eight recombinant small molecule collagens were effectively secreted and expressed in the supernatant, and the electrophoretic bands were single. There was no problem with the size of the hydrolyzed small molecule collagen being uncontrollable (collagen has a certain electrophoretic migration delay during electrophoresis, so its apparent molecular weight will be larger during electrophoresis).

[0144] Eight kinds of high-purity recombinant small molecule collagen freeze-dried products were taken for LC-MS analysis to obtain their deconvolution molecular weights (commissioned by Beijing Biotech Biotechnology Co., Ltd.). The results are shown in Figures 2 to 5. In some of the result figures, the deconvolution molecular weight values ​​are displayed with one decimal place and are rounded off. As can be seen in the figure, the theoretical molecular weight of 3A5D1NT is 8596.08Da, and the measured deconvolution molecular weight is 8796.68Da; the theoretical molecular weight of 3A5D1 is 9281.78Da, and the measured deconvolution molecular weight is 9333.96Da; the theoretical molecular weight of 3A5D2NT is 4532.83Da, and the measured deconvolution molecular weight is 4591.46Da; the theoretical molecular weight of 3A5D2 is 5218.54Da, and the measured deconvolution molecular weight is 5173.04Da; the theoretical molecular weight of 17S1NNT is 7483.28Da, and the measured deconvolution molecular weight is 764 8.41Da; the theoretical molecular weight of 17S1N is 8306.13Da, and the measured deconvolution molecular weight is 8306.00Da; the theoretical molecular weight of 17S3NT is 5406.08Da, and the measured deconvolution molecular weight is 5499.72Da; the theoretical molecular weight of 17S3 is 6228.93Da, and the measured deconvolution molecular weight is 6503.79Da; the error is caused by the glycosylation modification that may occur in the expression process of the protein and the detection error. Therefore, the measured deconvolution molecular weights of the recombinant small molecule collagen 3A5D1NT, 3A5D1, 3A5D2NT, 3A5D2, 17S1NNT, 17S1N, 17S3NT, and 17S3 of the present invention are basically consistent with the theoretical values.

[0145] Because 3A5D1, 3A5D2, 17S1N, and 17S3 have a 6×His tag at their carboxyl termini, Western blot analysis was performed using an anti-6×His Tag antibody (purchased from Nanjing GenScript Biotech Co., Ltd.). ECL chemiluminescence was used for development, and detection was performed using an automated chemiluminescence image analysis system (Tanon 5200). The results are shown in Figure 6 . The 6×His tag was successfully detected in 3A5D1, 3A5D2, 17S1N, and 17S3 (the negative control (-) was culture supernatant from the GS115 null strain, and the positive control (+) was His-tagged recombinant human MIF, purchased from Sangon Biotech (Shanghai) Co., Ltd.; both were normal). The corresponding band positions were consistent with the expected sizes.

[0146] The lyophilized product was enzymatically digested with trypsin, and the peptides of the recombinant collagen after trypsin digestion were detected by Nano-HPLC-MS / MS mass spectrometry (commissioned by Suzhou Putai Biotechnology Co., Ltd.). The detected peptides were then compared with the natural protein sequences in the Uniprot database. The results are shown in Figures 7-10. The mass spectrometry results show that the peptides obtained after enzymatic digestion of the recombinant small molecule collagens 3A5D1, 3A5D2, 17S1N, 17S3, 3A5D1NT, 3A5D2NT, 17S1NNT, and 17S3NT, and the sequences they cover, all belong to the relevant regions of the human collagen sequence, indicating that the recombinant small molecule collagen of the present invention was successfully expressed.

[0147] Example 2: Construction of a high expression system or high expression chassis cells or chassis engineering bacteria

[0148] (1) Design of CPB localization fusion functional protein

[0149] In the present invention, Pichia pastoris is used as the starting cell, and a special expression sequence design is used to first construct an exclusive chassis cell or chassis engineering bacteria. The chassis cell or chassis engineering bacteria includes a localized fusion functional protein. The localized fusion functional protein is used to cut and remove the C-terminal basic amino acid residues (K, R) of multiple tandemly repeated recombinant small molecule collagen proteins after they are cut into monomers by the Kex2 enzyme.

[0150] The localization fusion functional protein includes a CPB enzyme mature peptide sequence, a functional region sequence having intracellular membrane localization or conversion and transport functions between organelles, and a connecting sequence. The connecting sequence is used for connection when the CPB enzyme is fused with the functional region sequence having intracellular membrane localization or conversion and transport functions between organelles.

[0151] The CPB enzyme may be derived from any species, but is preferably derived from humans or rats.

[0152] The functional region sequence having the function of intracellular membrane localization or conversion and transport between organelles is preferably derived from Saccharomyces cerevisiae or Pichia pastoris. The functional region is preferably Kex2 enzyme, or other protein functional regions with similar functions, such as Ste13 protease.

[0153] The linker sequence is not limited, as long as it can connect the CPB enzyme without affecting the connection between the CPB enzyme and the functional region with intracellular membrane localization or conversion and transport between organelles.

[0154] In this embodiment, the CPB enzyme is preferably a human CPB enzyme full-length (including signal peptide, propeptide, mature peptide) sequence (Uniprot database ID is P15086, https: / / www.uniprot.org / uniprotkb / P15086) in which the amino acid sequence at positions 111-417 (i.e., the human CPB enzyme mature peptide sequence) is the preferred human CPB enzyme amino acid sequence in the present invention, as shown in SEQ ID NO. 17:

[0155] In this embodiment, the CPB enzyme is also preferably the amino acid sequence at positions 109-415 (i.e., the mature peptide sequence of the rat CPB enzyme) in the full-length sequence of the rat CPB enzyme (including signal peptide, propeptide, and mature peptide) (Uniprot database ID is P19223, https: / / www.uniprot.org / uniprotkb / P19223) as shown in SEQ ID NO. 18:

[0156] In this embodiment, the functional region having intracellular membrane localization or conversion and transport between organelles is preferably the amino acid sequence 679-814 of the full-length amino acid sequence of the Saccharomyces cerevisiae Kex2 enzyme (Uniprot database ID is P13134 protein, https: / / www.uniprot.org / uniprotkb / P13134), that is, the region at the carboxyl terminus having intracellular membrane localization and conversion and transport between organelles, the sequence of which is shown in SEQ ID NO. 19:

[0157] In this embodiment, the functional region having the function of intracellular membrane localization or conversion and transport between organelles is preferably the amino acid sequence 681-777 of the full-length amino acid sequence of the Kex2 enzyme of Komagataella phaffii (Pichia pastoris) (Uniprot database ID is protein C4R095, https: / / www.uniprot.org / uniprotkb / C4R095), i.e., the region at the carboxyl terminus having the function of intracellular membrane localization and conversion and transport between organelles, as shown in SEQ ID NO. 20:

[0158] As a linker sequence for the fusion expression of CPB enzyme and Kex2 enzyme carboxyl terminal sequence, a linker sequence is used, as shown in SEQ ID NO.21: GGSGSGSGGS

[0159] The CPB localization fusion functional protein constructed by the present invention is as follows:

[0160] The CPB localization fusion functional protein constructed by the present invention, which is a fusion expression of human CPB enzyme and the carboxyl terminal sequence of Pichia pastoris Kex2 enzyme, is named HCPB-PPKEX2, and its sequence is shown in SEQ ID NO.22:

[0161] The DNA sequence encoding SEQ ID NO.22 is shown in SEQ ID NO.23:

[0162] The CPB localization fusion functional protein of the fusion expression of human CPB enzyme and the carboxyl terminal sequence of Saccharomyces cerevisiae Kex2 enzyme is named HCPB-SCKEX2, and its sequence is shown in SEQ ID NO.24:

[0163] The DNA sequence encoding SEQ ID NO.24 is shown in SEQ ID NO.25:

[0164] The CPB localization fusion functional protein of the rat CPB enzyme and the carboxyl terminal sequence of the Pichia pastoris Kex2 enzyme was fused and expressed, and was named RCPB-PPKEX2. The sequence thereof is shown in SEQ ID NO.26:

[0165] The DNA sequence encoding SEQ ID NO.26 is shown in SEQ ID NO.27:

[0166] The CPB localization fusion functional protein of the rat CPB enzyme and the carboxyl terminal sequence of the Saccharomyces cerevisiae Kex2 enzyme was fused and expressed, and was named RCPB-SCKEX2. The sequence thereof is shown in SEQ ID NO.28:

[0167] The DNA sequence encoding SEQ ID NO.28 is shown in SEQ ID NO.29:

[0168] In the design of the present invention's targeted fusion functional protein, the preferred enzyme, Kex2, is a calcium-dependent serine protease expressed by yeast (including Pichia pastoris). It specifically recognizes and cleaves carboxyl-terminal peptide bonds of dibasic amino acids such as RR and KR within amino acid sequences, playing a key role in the yeast protein secretion pathway. Yeast also harbor the STE13 gene, which expresses the Ste13 protease (strictly speaking, a dipeptidyl aminopeptidase) intracellularly, capable of cleaving amino acid sequences such as EA and EAEA at the amino termini of proteins.

[0169] The Kex2 enzyme is most commonly used in genetic engineering in the yeast exogenous secretion pathway to cleave signal peptides or propeptide sequences in exogenous protein precursors, releasing the mature secretory protein. Kex2 cleaves the peptide bond at the carboxyl termini of the exogenous protein's KR or RR double base amino acid residues. To improve cleavage efficiency, amino acid sequences such as EA and EAEA are often added after KR or RR (optional; sequences of the exogenous protein can also be used, but cleavage efficiency varies depending on the 1-4 amino acid residues following KR or RR). Kex2 can also serve as a cleavage site between fusion proteins, cleaving a single expressed peptide into two or more fragments. This is particularly useful when expressing proteins with different subunits. Similarly, this application can also be applied to the expression of multiple protein sequences in tandem, where the addition of a Kex2 cleavage site can indirectly increase the copy number and thereby enhance the expression of the exogenous protein. However, when Kex2 is activated, dibasic amino acids such as KR and RR are a prerequisite for effective cleavage. However, KR and RR cannot be removed after cleavage. To improve cleavage efficiency, 1-2 amino acid residues are often added before KR and RR (optional and can be adjusted and designed based on actual conditions). For recombinantly expressed proteins, this introduces more non-self amino acids. Collagen, on the other hand, typically has a repeating triplet structure of GXY. The introduction of non-self amino acids often disrupts this structural feature.

[0170] During the design of the localization fusion protein of the present invention, the removal of KR and RR needs to be considered.

[0171] Recombinant carboxypeptidase B (CPB enzyme, an exocrine protease) can specifically cleave basic amino acids (especially K and R) at the carboxyl terminus of proteins until all the basic amino acids at the C-terminus are cleaved, leaving other non-basic amino acids exposed at the C-terminus of the protein. CPB enzymes are commonly used in the fields of bioengineering or biopharmaceuticals. Basically, after the recombinant protein or protein drug is expressed and purified, CPB enzyme is added to remove basic amino acids such as K and R at the C-terminus of the protein. This is equivalent to adding an exogenous substance to the expression and purification of the recombinant protein. In addition to the increased cost of purchasing CPB enzyme, the process of CPB removal and residual detection must also be added. If necessary, the residual CPB enzyme must be evaluated accordingly. The difficulty, process, cost, and time of the entire process will also increase. There is also the hidden danger of residual CPB enzyme due to incomplete removal.

[0172] Therefore, the present invention also requires establishing a synthetic metabolic pathway for the CPB enzyme within the cell, especially within the protein expression and secretion pathway, so that the CPB enzyme is localized intracellularly (in the protein expression and secretion pathway composed of the endoplasmic reticulum lumen, the trans-Golgi network (TGN), etc.). After the Kex2 enzyme cleaves the designed long sequence of recombinant collagen amino acids into small fragments of low-molecular-weight proteins, the CPB enzyme can cooperate with the Ste 13 protease to remove non-collagenous amino acids such as KR and EA at the C-terminus and N-terminus, respectively, maintaining the collagen GXY triplet repeat structure.

[0173] In summary, the present invention aims to solve the problem of not only expressing the CPB enzyme intracellularly, but also to enable it to enter the secretory expression pathway (in a manner completely consistent with secretory expression) while still being retained in the protein secretion pathway such as the endoplasmic reticulum and the Golgi apparatus without being secreted extracellularly, and to maintain temporal and spatial continuity with the activation time of the Kex2 enzyme.

[0174] Therefore, the present invention fuses the CPB enzyme with a sequence that has the function of localization on the inner membrane or conversion and transport between secretory organelles in the cell to form a new CPB localization fusion functional protein, which can be secreted, localized on the inner membrane of the cell, and can also accompany the Kex2 enzyme and recombinant collagen in conversion and transport between secretory organelles in the cell, and is not secreted (located on the inner membrane of the cell and cannot detach).

[0175] The Kex2 enzyme as a whole includes seven parts: a signal peptide, a precursor peptide, a catalytic domain, a P domain, a Ser / Thr-rich domain, a transmembrane region, and an extracellular domain. Its enzymatic activity is mainly achieved by the catalytic domain, the P domain, and the Ser / Thr-rich domain. Retaining these three parts can still maintain the activity of the Kex2 enzyme, and the C-terminal membrane region and the extracellular domain are mainly responsible for the conversion and transport of the Kex2 protease between the various organelles in the cell. The present invention selects the relevant functional region of the C-terminal end of the Kex2 enzyme for fusion expression with the CPB enzyme. In order not to affect the structure and activity of the two fused parts, a flexible Linker sequence (optional) is added in the middle. Moreover, the same C-terminal sequence will concentrate the Kex2 enzyme and the CPB localization fusion functional protein in the same area. The product of the Kex2 enzyme is the substrate of the CPB localization fusion functional protein, and concentrating in the same area is more conducive to the synergistic effect of the two enzymes.

[0176] (2) Construction and identification of expression vectors and chassis cells for CPB localization fusion functional proteins

[0177] CPB chassis cells need to express the CPB localization fusion functional protein and localize it in the cellular protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus, where it can synergize with yeast's own proteases such as Kex2 and Ste13. Therefore, when constructing an expression vector for the CPB localization fusion functional protein, a signal peptide sequence needs to be added to enable its secretion into the endoplasmic reticulum and subsequent protein secretion pathways. Signal peptides used for proteins that can enter the endoplasmic reticulum lumen have similar functions. The commonly used α-factor secretion signal is used as an example of the signal peptide sequence in this invention.

[0178] There are many expression vectors that can be used in Pichia pastoris. The promoter contained in the vector plays an important role. Commonly used promoters include: (1) inducible promoters such as AOX1 promoter, FLD1 promoter, FDH1 promoter, DAS1 promoter and DAS2 promoter. Methanol or methylamine can be used to induce protein expression. The addition of inducers can be used to regulate the timing of transcriptional expression of CPB localization fusion functional protein; (2) constitutive promoters such as GAP and GCW14 that do not rely on inducers. As long as the cell is in a viable state, they will start the transcriptional expression of the protein, that is, CPB localization fusion functional protein. Functional proteins are always transcribed and expressed; (3) THI4 promoter, THI11 and other promoters that do not require specific inducers can start protein expression by reducing some culture substances (such as reducing thiamine), and can also regulate the timing of transcriptional expression of CPB localization fusion functional proteins to a certain extent; (4) Promoters that regulate other conditions of the culture environment, such as the HSP82 promoter, are heat-inducible promoters. When the strain grows normally, changing the growth temperature and performing heat stimulation can induce the HSP82 promoter to start transcription, thereby regulating the transcriptional expression of CPB localization fusion functional proteins. However, no matter which promoter is used, the ultimate effect is the same, that is, to start the transcriptional expression of CPB localization fusion functional proteins. There is no difference in the ultimate effect, and all are within the scope of protection of the present invention. The present invention uses the common AOX1 promoter as an example. There are many expression vectors for Pichia pastoris or yeast. As long as the ultimate goal is to complete the expression of CPB localization fusion functional proteins, their effects are the same. The present invention uses commonly used expression vectors to construct multiple expression vectors.

[0179] DNA sequences SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, and SEQ ID NO.29 were synthesized by Nanjing GenScript Biotechnology Co., Ltd. The synthesized genes were cloned into expression vectors such as pPICZαB, pFLDα, and pPIC9K (all purchased from Thermo Fisher Scientific (China) Co., Ltd.) respectively. The cloning position was between the kex2 restriction site sequence in the α-factor secretion signal sequence (its terminal DNA sequence is AAAGAAGAAGGGGTATCTCTCGAGAAAAGA) and the termination codon "TGA" on the vector. Three sets of recombinant expression vectors required for the construction of chassis microorganisms are available:

[0180] (1) pPICZαB-HCPB-PPKEX2, pPICZαB-HCPB-SCKEX2, pPICZαB-RCPB-PPKEX2, and pPICZαB-RCPB-SCKEX2, using the AOX1 promoter and the α-factor secretion signal peptide sequence;

[0181] (2) pFLDα-HCPB-PPKEX2, pFLDα-HCPB-SCKEX2, pFLDα-RCPB-PPKEX2, and pFLDα-RCPB-SCKEX2, using the FLD promoter and the α-factor secretion signal peptide sequence;

[0182] (3) pPIC9K-HCPB-PPKEX2, pPIC9K-HCPB-SCKEX2, pPIC9K-RCPB-PPKEX2, and pPIC9K-RCPB-SCKEX2, using the AOX1 promoter and α-factor secretion signal peptide sequence.

[0183] Each of these three groups of recombinant expression vectors can be used alone, and all use the α-factor secretion signal peptide sequence. Although the promoters are different, they can all be induced using methanol (the FLD promoter can also use methylamine), and the final effect is the same. In the examples of the present invention, the first group is used as an example for the subsequent construction of engineering strains, as shown in Figures 11 to 14, which are four of the recombinant expression vectors constructed in this embodiment.

[0184] The blank starting strain used for the chassis cell can be the original Pichia pastoris strain or commercial strains such as X-33, SMD1168, GS115, or other yeasts. The final effect achieved is the same, that is, the expression of the CPB localization fusion functional protein is constructed in the cell protein secretion pathway, and synergistically acts with the yeast's own Kex2, Ste13 and other proteases to express and split the long protein. The present invention uses the commercial blank starting strain GS115 as an example.

[0185] The pPICZαB-HCPB-PPKEX2, pPICZαB-HCPB-SCKEX2, pPICZαB-RCPB-PPKEX2, and pPICZαB-RCPB-SCKEX2 vector plasmids were digested with Pme I (purchased from Dalian TaKaRa Company) at 37°C overnight to linearize them. The linearized plasmids were then recovered using a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to control the volume to about 10 μL. The linearized plasmids were electroporated into the competent cells of the empty host strain Pichia pastoris GS115 (purchased from the China Industrial Microbiological Culture Collection) and spread onto YPD plates containing 0.1 mg / mL Zeocin (purchased from Thermo Fisher Scientific (China) Co., Ltd.). After single colonies grew, single colonies were picked with a toothpick and inoculated into 96-well plates. After culturing at 30°C for 48 hours, the plates were spotted on plates containing 0.5 mg / mL and 1 mg / mL Zeocin, respectively. Zeocin YPD plates were selected, and strains growing on high concentration (1 mg / mL) Zeocin plates were inoculated into BMGY medium shake flasks. After overnight culture at 30°C and 220 rpm, the medium was replaced with BMMY medium for induced expression. 100% methanol was supplemented every 24 hours to a final concentration of 1.0%. The culture was cultured for more than 36 hours, and the culture supernatant and bacteria were collected. The bacteria were lysed with 8M urea, and the samples were prepared for SDS-PAGE detection. Antibodies against human or mouse CPB enzyme (purchased from Sangon Biotech (Shanghai) Co., Ltd.) were used, and recombinant human or rat CPB enzyme protein (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was used as a positive control. The culture supernatant of the empty host bacteria was used as a negative control. ECL chemiluminescence was used for color development, and the results were detected and synthesized using an automatic chemiluminescence image analysis system (Tanon 5200).

[0186] The results are shown in Figure 15, which shows the detection results of the expression of CPB localization fusion functional proteins HCPB-PPKEX2, HCPB-SCKEX2, RCPB-PPKEX2, and RCPB-SCKEX2 in intracellular lysate and culture supernatant; lanes in the left figure, 1: HCPB-SCKEX2 bacterial lysate; 2: HCPB-SCKEX2 bacterial culture supernatant; 3, 6: HCPB-PPKEX2 culture supernatant; 4, 5: HCPB-PPKEX2 bacterial lysate; (-): negative control; (+): positive control, recombinant human CPB enzyme; / : well with no sample. Lanes in the right figure, (-): negative control; (+): positive control, recombinant rat CPB enzyme; / : well without sample; 7: RCPB-SCKEX2 bacterial culture supernatant; 8: RCPB-SCKEX2 bacterial lysate; 9, 10, 12, 13: RCPB-PPKEX2 culture supernatant; 11, 14: RCPB-PPKEX2 bacterial lysate.

[0187] Human CPB enzyme, used as a positive control, was successfully detected. HCPB-PPKEX2, HCPB-SCKEX2, RCPB-PPKEX2, and RCPB-SCKEX2 were all detected only in intracellular lysates. The bands were larger than those of the positive control, and the band sizes were consistent with expectations for fusion proteins (when fused with other proteins, the apparent molecular weight on electrophoresis will naturally be larger). Most importantly, no corresponding bands were detected in the culture supernatant, indicating that HCPB-PPKEX2, HCPB-SCKEX2, RCPB-PPKEX2, and RCPB-SCKEX2 expressed as fusion proteins were not secreted extracellularly, but were all located intracellularly. This is consistent with the design purpose of the four CPB localization fusion functional proteins, namely, the human or rat CPB enzyme is fused with the sequence of the inner membrane localization and conversion and transport function region between organelles derived from the carboxyl terminus of the Kex2 enzyme. Under the action of the signal peptide, they can enter the intracellular protein secretion pathway composed of the endoplasmic reticulum and the Golgi apparatus, but like the Kex2 enzyme, they can only exist inside the cell and cannot be secreted outside the cell.

[0188] Referring to the Invitrogen operating manual, the YPD plate gradient with different concentrations of Zeocin antibiotics and the spot plate screening method were used, combined with the intracellular WB detection results, to successively screen out four engineered strains of chassis cells fused with functional protein synthesis metabolic pathways: HCPB-PPKEX2, HCPB-SCKEX2, RCPB-PPKEX2, and RCPB-SCKEX2, as host bacteria for subsequent recombinant tandem expression strategies.

[0189] The engineered strain samples of the four chassis cells that constructed the CPB localization fusion protein functional pathway were sent to the General Microbiology Center of the China Microorganism Culture Collection Administration. The strain collection numbers correspond to:

[0190] The strain expressing HCPB-PPKEX2 has the following deposit number: CGMCC No. 25815;

[0191] The strain expressing RCPB-PPKEX2 has the deposit number: CGMCC No. 25817;

[0192] The strain expressing HCPB-SCKEX2 has the deposit number: CGMCC No. 25816;

[0193] The strain expressing RCPB-SCKEX2 has the deposit number: CGMCC No. 25818;

[0194] The deposit address of the above-mentioned engineered bacteria is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the deposit date is: September 26, 2022. The taxonomic name is: Pichia pastoris Komagataella phaffii.

[0195] Example 3: Design of small molecule collagen amino acid sequence and characterization of small molecule collagen expressed in tandem repeats

[0196] In order to achieve the purpose of efficient expression of the small molecule collagen described in the present invention, the amino acid sequence of the target small molecule collagen was designed in the present invention. The design requirements are:

[0197] (1) The sites that can be effectively recognized and cut by yeast Kex2 enzyme are designed between the repeated series of small molecule collagen monomers (or fragments), and their prominent features are the presence of double basic amino acids such as KR and RR.

[0198] (2) Following the dibasic amino acid residue may be EA, EAEA or other amino acid sequences that are helpful for Kex2 enzyme recognition and cleavage, such as D or A, or directly the amino acids of the small molecule collagen protein monomer (or fragment) itself (there are relevant examples in the invention).

[0199] (3) The amino acid sequence of the repeated tandem small molecule collagen monomer (or fragment) can be any sequence. The specific order of the amino acid residues, the sequence length, and the number of tandem repetitions of the monomer (or fragment) can be freely arranged and are not restricted. Of course, as a small molecule collagen, it generally needs to have a typical GXY triplet structure, and the amino acid sequence length is controlled within 100 amino acids.

[0200] The present invention is exemplified as follows:

[0201] (1) With reference to the amino acid sequence of 3A5D2NT, a tandem repeat sequence was designed, repeated 9 times and named 3A5D29N-9, with a total of 522 amino acids. Its sequence is shown in SEQ ID NO. 30:

[0202] When 3A5D29N-9 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into the same small molecule protein sequence between the KR and EA in each KREA, the sequence of which is shown in SEQ ID NO.31:

[0203] The two amino acids EA at the amino terminus are cleaved and removed by Ste 13 protease, and the two amino acids KR at the carboxyl terminus are cleaved and removed by CPB enzyme. The final secreted product is a 54-amino acid recombinant small molecule collagen (denoted as 3A5D29N). The amino acid sequence is a typical GXY triplet collagen sequence, as shown in SEQ ID NO.32:

[0204] (2) With reference to the amino acid sequence of 17S3NT, a tandem repeat sequence was designed, repeated 8 times, named 17S28-8, with a total of 536 amino acids, and its sequence is shown in SEQ ID NO. 33:

[0205] When 17S28-8 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into the same small molecule protein sequence between the KR and EA in each KREA, the sequence of which is shown in SEQ ID NO.34:

[0206] The two amino acids EA at the amino terminal are cleaved and removed by Ste 13 protease, and the two amino acids KR at the carboxyl terminal are cleaved and removed by CPB enzyme. The final secreted product is a 63-mer (denoted as 17S28), and the amino acid sequence is a typical GXY triplet collagen sequence, as shown in SEQ ID NO.35:

[0207] (3) Referring to the amino acid sequence of 17S1NNT, a tandem repeat sequence was designed, repeated 6 times, named 17S1N6-6, with a total of 540 amino acids, and its sequence is shown in SEQ ID NO. 36:

[0208] When 17S1N6-6 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into the same small molecule protein sequence between the KR and EA in each KREA, the sequence of which is shown in SEQ ID NO.37:

[0209] The two amino acids EA at the amino terminal are cleaved and removed by Ste 13 protease, and the two amino acids KR at the carboxyl terminal are cleaved and removed by CPB enzyme. The final secreted product is a segment of 86 (denoted as 17S1N6), and the amino acid sequence is a typical GXY triplet collagen sequence, as shown in SEQ ID NO.38:

[0210] (4) Referring to the amino acid sequence of 17S1NNT, a tandem repeat sequence was designed, repeated 7 times, named 17S1N7-7, with a total of 528 amino acids, and its sequence is shown in SEQ ID NO. 39:

[0211] When 17S1N7-7 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into the same small molecule protein sequence between the KR and EA in each KREA, the sequence of which is shown in SEQ ID NO.40:

[0212] The two amino acids EA at the amino terminal are cleaved and removed by Ste 13 protease, and the two amino acids KR at the carboxyl terminal are cleaved and removed by CPB enzyme. The final secreted product is a segment of 84 (denoted as 17S1N7), the sequence of which is shown in SEQ ID NO.41:

[0213] (5) Referring to the amino acid sequence of 17S1NNT, a tandem repeat sequence was designed and repeated 7 times, named 17S1NK-7, with a total of 595 amino acids, and its sequence is shown in SEQ ID NO. 42:

[0214] When 17S1NK-7 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into the same small molecule protein sequence between the KR and EA in each KREA, the sequence of which is shown in SEQ ID NO.43:

[0215] The two amino acids EA at the amino terminus are cleaved and removed by Ste 13 protease, and the two amino acids KR at the carboxyl terminus are cleaved and removed by CPB enzyme. The final secreted product is a recombinant small molecule collagen protein of 84 amino acids (denoted as 17S1NK), the sequence of which is shown in SEQ ID NO.44:

[0216] (6) Referring to the amino acid sequence of 3A5D1, a tandem repeat sequence was designed and repeated five times, named 3A5D15D-5, with a total of 510 amino acids, and its sequence is shown in SEQ ID NO. 45:

[0217] When 3A5D15D-5 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into the same small molecule protein sequence between the KR and D in each KRD, the sequence of which is shown in SEQ ID NO.46:

[0218] The two amino acids KR at the carboxyl end are cleaved and removed by CPB enzyme, and the final secreted product is a 100-amino acid recombinant small molecule collagen (denoted as 3A5D15D), which is a typical GXY triplet collagen sequence, as shown in SEQ ID NO.47:

[0219] (7) Similarly, the use of other restriction enzyme sites can also achieve the same effect as 3A5D15D. The restriction enzyme site region sequence is designed as KREA, and a repeated tandem sequence is designed and repeated 5 times, named 3A5D15E-5, with a total of 520 amino acids, and its sequence is shown in SEQ ID NO. 48:

[0220] When 3A5D15E-5 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into the same small molecule protein sequence between the KR and EA in each KRD, the sequence of which is shown in SEQ ID NO.49:

[0221] The two amino acids EA at the amino terminus are cleaved and removed by Ste 13 protease, and the two amino acids KR at the carboxyl terminus are cleaved and removed by CPB enzyme. The final secreted protein is a 100-amino acid recombinant small molecule collagen (the same sequence as that obtained after expression and cleavage of 3A5D15D, here referred to as 3A5D15E), which is a typical GXY triplet collagen sequence, as shown in SEQ ID NO. 50:

[0222] (8) Referring to the amino acid sequence of 3A5D1NT, a tandem repeat sequence was designed and repeated 6 times, named 3A5D15R-6, with a total of 570 amino acids, and its sequence is shown in SEQ ID NO. 51:

[0223] When 3A5D15R-6 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into the same small molecule protein sequence between the KR and D in each KRD, the sequence of which is shown in SEQ ID NO.52:

[0224] The two amino acids KR at the carboxyl end are cleaved and removed by CPB enzyme, and the final secreted product is a 93-amino acid recombinant small molecule collagen (denoted as 3A5D15R), the sequence of which is shown in SEQ ID NO.53:

[0225] (9) Referring to the amino acid sequence of 3A5D1NT, a tandem repeat sequence was designed and repeated 6 times, named 3A5D15KR-6, with a total of 582 amino acids, and its sequence is shown in SEQ ID NO. 54:

[0226] When 3A5D15KR-6 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into the same small molecule protein sequence between the KR and D in each KRD, the sequence of which is shown in SEQ ID NO.55:

[0227] The two amino acids KR at the carboxyl end are cleaved and removed by CPB enzyme, and the final secreted product is a 95-amino acid recombinant small molecule collagen (denoted as 3A5D15KR), the sequence of which is shown in SEQ ID NO.56:

[0228] (10) Referring to the amino acid sequence of 3A5D1NT, a tandem repeat sequence was designed and repeated 6 times, named 3A5D15EKR-6, with a total of 576 amino acids, and its sequence is shown in SEQ ID NO. 57:

[0229] When 3A5D15EKR-6 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into the same small molecule protein sequence between the KR and D in each KRD, the sequence of which is shown in SEQ ID NO.58:

[0230] The two amino acids KR at the carboxyl end are cleaved and removed by CPB enzyme, and the final secreted product is a 94-amino acid recombinant small molecule collagen (denoted as 3A5D15EKR), which is a typical GXY triplet collagen sequence, as shown in SEQ ID NO.59:

[0231] (11) Referring to the amino acid sequence of 3A5D1NT, a tandem repeat sequence was designed and repeated 6 times, named 3A5D15G-6, with a total of 576 amino acids, and its sequence is shown in SEQ ID NO. 60:

[0232] When 3A5D15G-6 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into the same small molecule protein sequence between the KR and D in each KRD, the sequence of which is shown in SEQ ID NO.61:

[0233] The two amino acids KR at the carboxyl end are cleaved and removed by CPB enzyme, and the final secreted product is a 94-amino acid recombinant small molecule collagen (denoted as 3A5D15G), which is a typical GXY triplet collagen sequence, as shown in SEQ ID NO.62:

[0234] In addition to the design of repeated tandem sequences based on a region in the human type III collagen sequence or the human XVII collagen sequence as described above, sequences spliced ​​together from two or more regions of the human collagen sequence as monomers for tandem repetition can also be applied to the repeated tandem expression system (including the repeated tandem expression sequence design method and the use of chassis cells) and the monomer expression system described in the present invention.

[0235] (12) With reference to several different functional regions of the human type III collagen sequence (referenced to the protein sequence with ID P02461 in the Uniprot database, https: / / www.uniprot.org / uniprotkb / P02461), the amino acid sequences at positions 880-915, 949-966, 1012-1038, and 1060-1074 were combined to design a recombinant small molecule collagen, totaling 96 amino acids, which can be used for a tandem monomer. The sequence is shown in SEQ ID NO. 63.

[0236] Based on this, a tandem repeat sequence was designed, repeated 6 times, named 3A5D16M-6, with a total of 500 amino acids, and its sequence is shown in SEQ ID NO.64:

[0237] When 3A5D16M-6 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into the same small molecule protein sequence between the KR and EA in each KREA, the sequence of which is shown in SEQ ID NO.65:

[0238] The two amino acids EA at the amino terminus are cleaved and removed by Ste 13 protease, and the two amino acids KR at the carboxyl terminus are cleaved and removed by CPB enzyme. The final secreted product is a 96-amino acid recombinant small molecule collagen protein named 3A5D16M, whose sequence is shown in SEQ ID NO.66:

[0239] (13) With reference to the human type III collagen sequence (referenced to the protein sequence with ID P02461 in the Uniprot database, https: / / www.uniprot.org / uniprotkb / P02461), two functional regions, namely the amino acid sequence at positions 1060-1074 and the amino acid sequence at positions 1015-1038, totaling 39 amino acids, can be used for monomer expression, and the sequence is shown in SEQ ID NO. 67.

[0240] Based on this, a tandem repeat sequence was designed, repeated 12 times, named 3A5D15M-12, with a total of 516 amino acids, and its sequence is shown in SEQ ID NO.68:

[0241] When 3A5D15M-12 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into the same small molecule protein sequence between the KR and EA in each KREA, the sequence of which is shown in SEQ ID NO.69:

[0242] The two amino acids EA at the amino terminus are cleaved and removed by Ste 13 protease, and the two amino acids KR at the carboxyl terminus are cleaved and removed by CPB enzyme. The final secreted product is a 39-amino acid recombinant small molecule collagen protein named 3A5D15M, whose sequence is shown in SEQ ID NO.70:

[0243] In theory, artificially designed non-natural collagen sequences can also be applied to the repeated tandem expression system of the present invention. The principle is as described above and will not be repeated here.

[0244] Construction of recombinant expression vector of repeated tandem expression system and verification of expression effect:

[0245] In this embodiment, based on the design of the chassis cells and the aforementioned amino acid sequence, the expression vector used for the recombinant small molecule collagen is not limited. As long as the selected expression vector contains a signal peptide sequence that can direct the translated protein into the endoplasmic reticulum, commonly used expression vectors such as pPICZαB, pFLDα, and pPIC9K are suitable. Other constructions based on expression vectors with similar functions are also within the scope of protection of this invention. Similarly, the DNA sequence encoding the corresponding recombinant small molecule collagen can be optimized; as long as the final encoded amino acid sequence is the same, it is within the scope of protection of this invention.

[0246] The DNA sequence encoding 3A5D29N-9 is shown in SEQ ID NO.71:

[0247] The DNA sequence encoding 17S28-8 is shown in SEQ ID NO.72:

[0248] The DNA sequence encoding 17S1N6-6 is shown in SEQ ID NO.73:

[0249] The DNA sequence encoding 17S1N7-7 is shown in SEQ ID NO.74:

[0250] The DNA sequence encoding 17S1NK-7 is shown in SEQ ID NO.75:

[0251] The DNA sequence encoding 3A5D15D-5 is shown in SEQ ID NO.76:

[0252] The DNA sequence encoding 3A5D15E-5 is shown in SEQ ID NO.77:

[0253] The DNA sequence encoding 3A5D15R-6 is shown in SEQ ID NO.78:

[0254] The DNA sequence encoding 3A5D15KR-6 is shown in SEQ ID NO.79:

[0255] The DNA sequence encoding 3A5D15EKR-6 is shown in SEQ ID NO.80:

[0256] The DNA sequence encoding 3A5D15G-6 is shown in SEQ ID NO.81:

[0257] The DNA sequences SEQ ID NO. 71 to SEQ ID NO. 81 were synthesized by Nanjing GenScript Biotechnology Co., Ltd. The synthesized genes were cloned into the pPIC9K vector between bp 1203 (i.e., α-factor secretion signal / cleavage site 1203, search sequence AAAGAAGAAGGGGTATCTCTCGAGAAAAGA) and the restriction enzyme cleavage site Not I. Recombinant expression vectors pPIC9K-3A5D29N-9, pPIC9K-17S28-8, pPIC9K-17S1N6-6, pPIC9K-17S1N7-7, pPIC9K-17S1NK-7, pPIC9K-3A5D15D-5, pPIC9K-3A5D15E-5, pPIC9K-3A5D15G-6, pPIC9K-3A5D15EKR-6, pPIC9K-3A5D15KR-6, and pPIC9K-3A5D15R-6 were obtained. Other expression vectors that can be used in Pichia pastoris, such as pPICZαB and pFLDα, also have similar effects to pPIC9K.

[0258] 10 μg of the above-mentioned recombinant expression vector plasmids (pPIC9K-3A5D29N-9, pPIC9K-17S28-8, pPIC9K-17S1N6-6, pPIC9K-17S1N7-7, pPIC9K-17S1NK-7, pPIC9K-3A5D15D-5, pPIC9K-3A5D15E-5, pPIC9K-3A5D15G-6, pPIC9K-3A5D15EKR-6, pPIC9K-3A5D15KR-6, pPIC9K-3A5D15R-6) were respectively digested with SacⅠ (purchased from Dalian TaKaRa Company) at 37°C overnight to linearize them, and then the linearized plasmids were recovered using a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to control the volume to about 10 μL.

[0259] The linearized plasmid was electrotransformed into the competent cells of the four chassis engineering strains (HCPB-PPKEX2 strain, deposit number CGMCC No. 25815; RCPB-PPKEX2 strain, deposit number CGMCC No. 25817; HCPB-SCKEX2 strain, deposit number CGMCC No. 25816; RCPB-SCKEX2 strain, deposit number CGMCC No. 25818) of the CPB localization fusion functional protein functional pathway constructed in Example 2 of the present invention. The electroporated bacterial solution was spread on an MD plate, with 100 μL to 200 μL spread on one plate, allowed to stand at room temperature for 10 min, and inverted cultured at 30° C. for 2-5 days until a single colony (positive transformant) appeared.

[0260] Add 2 mL of sterile double-distilled water to the surface of the MD plate, and then gently scrape off the His on the surface of the plate with a sterile triangular spreader. + Transformants were isolated and transferred to a 50 mL centrifuge tube. The bacterial suspension was diluted with sterile double-distilled water, and 105 cells were plated onto a YPD plate containing 0.5 mg / mL G418. The plate was inverted and incubated at 30°C for 3-4 days until single colonies appeared. Colonies were picked from the YPD plate and transferred to a sterile 96-well plate (200 μL YPD / well), mixed, and incubated at 30°C for 48 hours. The bacterial suspension in each well was mixed and 10 μL was transferred to a new sterile 96-well plate. The plate was incubated at 30°C for 24 hours, and this process was repeated once. After 24 hours, 1 μL of the solution was taken from the third 96-well plate and spotted onto YPD plates containing 1.0 mg / mL and 4 mg / mL G418, respectively. The culture was continued at 30°C for 96-120 hours. If the Pichia pastoris transformant can grow on a plate containing a high concentration of G418, it means that the transformant can express the exogenous gene with high efficiency. After this step of screening, a recombinant yeast engineered strain with high expression efficiency can be obtained.

[0261] The following description will be given using the chassis cell engineering strain expressing HCPB-PPKEX2 (deposit number CGMCC No. 25815) and the chassis cell engineering strain expressing RCPB-PPKEX2 (deposit number CGMCC No. 25817) as expression host cells.

[0262] The repeated tandem expression system of the present invention has constructed a variety of engineered strains expressing recombinant small molecule collagen. The engineered strains constructed using five chassis cell engineered strains expressing HCPB-PPKEX2 as expression host cells were sent to the General Microbiology Center of the China Culture Collection Administration for Microorganisms for preservation. The strain deposit numbers correspond to:

[0263] The strain expressing the recombinant small molecule collagen 3A5D29N has the deposit number: CGMCC No.25819;

[0264] The strain expressing recombinant small molecule collagen 17S28 has the deposit number: CGMCC No.25821;

[0265] The strain expressing the recombinant small molecule collagen 3A5D15D has the deposit number: CGMCC No.25827;

[0266] The strain expressing the recombinant small molecule collagen 17S1N6 has the deposit number: CGMCC No.25829;

[0267] The strain expressing the recombinant small molecule collagen 3A5D15E has a deposit number of CGMCC No. 25828.

[0268] The engineered strains constructed with two chassis cell engineering strains expressing RCPB-PPKEX2 as expression host cells were sent to the General Microbiology Center of China Culture Collection Administration for Microorganisms for preservation. The strain preservation numbers correspond to:

[0269] The strain expressing the recombinant small molecule collagen 3A5D29N has the deposit number: CGMCC No.25820;

[0270] The strain expressing recombinant small molecule collagen 17S28 has the deposit number: CGMCC No.25822;

[0271] The deposit address of the above engineering strains is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the deposit date is: September 26, 2022. The taxonomic name is: Pichia pastoris Komagataella phaffii.

[0272] Individual colonies grown on the plates with high G418 concentration were selected and placed in 100 mL flasks containing 10 mL of BMGY medium. Culture was performed at 28-30°C and 220 rpm until the OD600 reached 2-6 (16-18 h). The cells were harvested by centrifugation at 1500-3000 g for 5 min at room temperature, and resuspended in BMMY medium to an OD600 of approximately 2. The cells were then grown on a shaker at 28-30°C and 220 rpm for another 3 days. 100% methanol was added to the culture medium every 24 h to a final concentration of 1.0%. Bacterial liquid samples were taken at different time points (sampling was performed once every 24 hours after the start of induction), with a sampling volume of 1 mL, placed in a 1.5 mL EP tube, centrifuged at 12000 rpm for 2-3 min, the supernatant was collected, and 5× loading buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol) was added, and the supernatant was heated in a 100°C metal bath for 10 min for SDS-PAGE detection.

[0273] The results of SDS-PAGE detection are shown in Figures 16-19. The results of pPIC9K-3A5D29N-9, pPIC9K-17S28-8, pPIC9K-17S1N6-6, pPIC9K-17S1N7-7, pPIC9K-17S1NK-7, pPIC9K-3A5D15D-5, pPIC9K-3A5D15E-5, pPIC9K-3A5D15G-6, pPIC9K-3A5D15EKR-6, pPIC9K-3A5D15KR-7, and pPIC9K-3A5D15D-5. D15R-6 successfully and effectively expressed recombinant small-molecule collagen of corresponding sizes in both chassis cell engineering strains, and the expression was effectively secreted into the supernatant, with a single electrophoretic band. This demonstrates that the purpose of the tandem repeat amino acid sequence design of the small-molecule collagen protein of the present invention has been achieved: that is, to express a large-molecule protein (a tandem repeat structure of a small-molecule collagen monomer) with up to several hundred amino acid sequences. Ultimately, in the chassis cell engineering strain, a recombinant small-molecule collagen with the same sequence and a single band can be expressed (collagen has a certain electrophoretic migration delay during electrophoresis, so its apparent molecular weight will be larger during electrophoresis).

[0274] The resulting lyophilized products of the small-molecule collagen proteins 3A5D29N, 17S28, 3A5D15D, and 17S1N6 were digested with trypsin. The peptides from these tryptic hydrolysis reactions were detected by Nano-HPLC-MS / MS mass spectrometry (performed by Suzhou Putai Biotechnology Co., Ltd.). Sequences of the detected peptides were then aligned using the Uniprot database. As shown in Figures 20 and 21 , the mass spectrometry results indicate that the peptides detected after enzymatic hydrolysis of the recombinant small-molecule collagen proteins 3A5D29N, 17S28, 3A5D15D, and 17S1N6, as well as the sequences they cover, all fall within the relevant regions of the human collagen sequence selected during amino acid sequence design, indicating successful expression.

[0275] The lyophilized products of 3A5D29N and 17S28 were subjected to LC-MS analysis (capillary high performance liquid chromatograph Thermo Fisher Scientific Ultimate 3000, electrospray-quadrupole time-of-flight mass spectrometer AB SCIEX TripleTOF 5600Mass Spectrometer, chromatographic column ACQUITY UPLC Protein BEH C4 Column) to obtain their deconvoluted molecular weights (commissioned by Beijing Biotech Biotechnology Co., Ltd.). The results are shown in part of the results in Figure 22 (the deconvoluted molecular weight values ​​in the figure are rounded to one decimal place). The theoretical molecular weight of 3A5D29N is 4988.35Da, and the measured deconvoluted molecular weight is 4987.5Da; the theoretical molecular weight of 17S28 is 5861.60Da, and the measured deconvoluted molecular weight is 5833.92Da; due to the glycosylation modification that may occur during the expression process of the protein and the errors caused by the detection, the measured deconvoluted molecular weights of the recombinant small molecule collagens 3A5D29N and 17S28 are basically consistent with the theoretical values.

[0276] Verification of chassis cell engineering strain effects and expression systems:

[0277] In addition to cleaving long protein sequences into single, short, small-molecule collagens, the chassis cell engineering bacteria constructed by the present invention also have an important function of cleaving non-collagen amino acid sequences at both the amino and carboxyl ends of the cleaved single, short, small-molecule collagen monomers. The expression of 3A5D29N-9 and 17S28-8 in HCPB-PPKEX2 chassis cell engineering strains and RCPB-PPKEX2 chassis cell engineering strains was used as an example. The expressed recombinant small-molecule collagens 3A5D29N and 17S28 were tested at both the amino and carboxyl ends to verify whether the synergistic effect of Kex2, Ste 13, and CPB enzymes in the chassis cell engineering strains was established. Beijing Biotech Biotechnology Co., Ltd. was commissioned to conduct N-terminal (amino terminus) and C-terminal (carboxyl terminus) sequencing verification and LC-MS / MS-based protein full sequence analysis of the recombinant small molecule collagen freeze-dried products expressed by 3A5D29N-9 and 17S28-8 in the HCPB-PPKEX2 chassis cell engineering strain and RCPB-PPKEX2 chassis cell engineering strain, respectively.

[0278] N-terminal sequencing: The N-terminal sequence of the sample was analyzed using the Shimadzu fully automatic protein peptide sequencer (PPSQ-33A) (Edman degradation method): an appropriate amount of recombinant small molecule collagen freeze-dried product was dissolved, the sample solution was dropped onto a PVDF membrane, and placed in a reactor. After the reactor was assembled, it was placed in a fixed position on the instrument. The software PPSQ-30Analysis was used to set the following: sample name, sample number, number of test cycles, and method file. After the settings were completed, the test began. The raw data and spectra generated by the PPSQ-33A were identified by the PPSQ-30DataProcessing software to identify the peaks and export the corresponding spectra. After data analysis, the protein N-terminal sequence was determined.

[0279] The results showed that the N-terminal sequence of the recombinant small molecule collagen expressed by 3A5D29N-9 in HCPB-PPKEX2 chassis cell engineering strains and RCPB-PPKEX2 chassis cell engineering strains was NH2-Gly-Lys-Ser-Gly-Asp-Arg-Gly, that is, GKSGDRG, which was consistent with the theoretical sequence.

[0280] The N-terminal amino acid sequence of the recombinant small molecule collagen expressed by 17S28-8 in the HCPB-PPKEX2 chassis cell engineering strain and the RCPB-PPKEX2 chassis cell engineering strain was detected to be: NH2-Gly-Val-Pro-Gly-Ser-Val-Gly, that is, GVPGSVG, which is consistent with the theoretical sequence.

[0281] The N-terminal amino acid sequence of (GVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLE) is consistent.

[0282] C-terminal sequencing: An appropriate amount of recombinant small molecule collagen freeze-dried product sample was dissolved and then treated with trypsin and pepsin. The treated sample was then analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) to obtain a raw file of the original mass spectrometry result. The raw file was analyzed and matched by the Byonic software. The mass spectrometry data was then retrieved from the database to obtain the identification result. The secondary mass spectra of the detected C-terminal peptide are shown in Figures 23 and 24 below.

[0283] The results showed that: (1) the C-terminal sequence of the recombinant small molecule collagen expressed by 3A5D29N-9 in HCPB-PPKEX2 chassis cell engineering strain and RCPB-PPKEX2 chassis cell engineering strain was GHRGLE, which was consistent with the theoretical sequence.

[0284] (GKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGLE) is consistent with the C-terminal sequence of the detection of 17S28-8 expressing recombinant small molecule collagen in the HCPB-PPKEX2 chassis cell engineering strain. The detection C-terminal sequence of 17S28-8 expressing recombinant small molecule collagen in the RCPB-PPKEX2 chassis cell engineering strain is KGPMGPPGPKGDQGEKGPRGLE, both of which are consistent with the theoretical sequence

[0285] The C-terminus of (GVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLE) is consistent.

[0286] Protein full sequence analysis based on LC-MS / MS: Further, samples of the recombinant small molecule collagen freeze-dried product were treated with trypsin, chymotrypsin, pepsin, trypsin & Glu-C protease, and trypsin & Asp-N protease. The treated samples were then analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) to obtain a raw file of the original mass spectrometry results. After analysis by the Byonic software, the data was matched to obtain the results of full sequence verification. Comprehensive analysis of the test results showed that the total amino acid sequence coverage of the recombinant small molecule collagen freeze-dried product samples of 3A5D29N-9 and 17S28-8 expressed in the HCPB-PPKEX2 chassis cell engineering strain and the RCPB-PPKEX2 chassis cell engineering strain, respectively, was 100%. The amino acid sequence of the small molecule collagen expressed by the present invention is consistent with the amino acid sequence of the target small molecule collagen.

[0287] In this example, when 3A5D29N-9 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into monomers with the same small molecule protein sequence:

[0288] The two amino acids EA at the amino terminus are cleaved and removed by Ste 13 protease, and the two amino acids KR at the carboxyl terminus are cleaved and removed by CPB enzyme. The final secreted product is a 54-amino acid recombinant small molecule collagen 3A5D29N:

[0289] In this example, when 17S28-8 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it into monomers with the same small protein sequence:

[0290] EAGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLEKR. The two amino acids EA at the amino terminus are removed by Ste 13 protease, and the two amino acids KR at the carboxyl terminus are removed by CPB enzyme. The final secreted product is a 63-amino acid recombinant small molecule collagen 17S28:

[0291] Whether the amino acid sequence of the N and C terminals is correct directly indicates whether the anabolic pathway of the CPB enzyme in the chassis cell engineering strain is effectively established, whether it has biological activity after establishment, and how it synergizes with its own yeast Kex2 enzyme and Ste13 protease. The results of this example show that the CPB enzyme in the chassis cell engineering strain (whether it is human CPB enzyme or rat CPB enzyme) has successfully established an anabolic pathway in Pichia pastoris and can normally exert its biological enzyme activity, and synergizes with the yeast's own Kex2 enzyme and Ste13 protease to successfully cut the long sequence of macromolecular recombinant collagen designed with specific amino acids after expression and remove the non-collagenous sequences at both ends to secrete recombinant small molecule collagen.

[0292] Example 4: 500L production verification of small molecule collagen

[0293] Small fermenters or shake flasks are unlikely to demonstrate the practicalities of large-scale industrial production. Only 500L fermenters offer the potential for scale-up and demonstrate the practicalities of large-scale industrial production. This study optimized the fermentation process for high-expression engineered strains and verified the yield of each engineered strain in a 500L fermenter.

[0294] The two recombinant small molecule collagens 3A5D2NT and 3A5D29N are highly similar in length, highly homologous in sequence, and have the same purification method. The present invention specifically compares the expression of small molecule collagens using these two strains.

[0295] As shown in the table below, two engineering bacteria (the engineering strain expressing 3A5D29N is CGMCC No. 25819, and the engineering strain expressing 3A5D2NT is CGMCC After optimization of fermentation parameters for strain No. 25812, the corresponding indicators were essentially consistent. However, there were significant differences in the collagen expression in the fermentation supernatant (UV method, UV protein quantification empirical formula: C (mg / mL) = 0.144 * (A215 - A225) for determining protein concentration) and the yield of the final purified lyophilized product. The expression level of 3A5D29N in the fermentation supernatant was generally about twice that of 3A5D2NT (the UV method was significantly affected by pigments in the fermentation broth, resulting in a low multiple ratio), and the yield of the final purified lyophilized product was 4-5 times that of 3A5D2NT (directly weighed). In the optimal batch fermentation, the protein yield in the fermentation supernatant (UV method) of 3A5D29N was twice that of 3A5D2NT. The SDS-PAGE analysis of the fermentation supernatant is shown in Figure 25. When comparing the yields of the purified lyophilized products obtained from the same volume of fermentation broth, the yield of 3A5D29N was nearly 5 times that of 3A5D2NT (directly weighed), representing a significant improvement in yield. The yield of recombinant small molecule collagen in other recombinant small molecule collagen repeat tandem expression systems is also 4 to 6 times that of the small molecule collagen monomer sequence expression.

[0296] Example 5: Biological activity and transdermal absorption detection of recombinant small molecule collagen

[0297] (1) Cell adhesion and viability assay

[0298] The cell adhesion activity detection method of recombinant small molecule collagen refers to Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura. Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649 (2004).

[0299] Specific implementation method: Normally culture NIH / 3T3 cells (purchased from the cell bank of the Chinese Academy of Sciences, product number GNM6, culture and passaging methods are carried out according to the cell instructions). Take the recombinant small molecule collagen 3A5D29N, 3A5D2NT, 3A5D1NT, 17S1NNT, 17S3NT, 17S28 freeze-dried products, and compare them with the recombinant type III collagen (patent application number CN201310033299.6, published on May 15, 2013, molecular weight 43.6KDa) and recombinant type XVII collagen freeze-dried sponge (patent application number CN202110520499.9, published on July 30, 2021, molecular weight 23.8KDa) produced by our company (Jiangsu Chuangjian Medical Biotechnology Co., Ltd.), and compare natural human collagen (Sigma, product number C7774) and bovine serum albumin (BSA, purchased from Sangon Biotech (Shanghai) Co., Ltd.) and dissolve (ultrapure water or 1M HCl solution), and the protein concentration was determined using the UV protein quantitative empirical formula: C (mg / mL) = 0.144*(A215-A225), and then diluted to 0.5 mg / mL with PBS (pH 7.4).

[0300] 100 μL of each protein solution and a blank PBS solution were added to a 96-well cell culture plate and incubated at room temperature for 60 minutes. 105 well-cultured NIH / 3T3 cells were then added to each well and incubated at 37°C, 5% CO2 for 60 minutes. The cells were washed four times with PBS. The absorbance at OD492 nm was measured using an LDH assay kit (Roche, 04744926001). Data were analyzed and significant differences were analyzed (SPSS 22 software, Ducan method, P < 0.05).

[0301] The absorbance at OD492nm reflects the cell adhesion activity of the collagen sample: higher adhesion activity indicates more cells adhere to the protein. Collagen can quickly help cells adhere to the wall or extracellular matrix, thereby promoting the creation of an optimal extracellular environment. As shown in Figure 26, the cell adhesion activities of the recombinant low-molecule collagens 3A5D29N, 3A5D2NT, 3A5D1NT, 17S1NNT, 17S3NT, and 17S28 are comparable to or superior to those of native human collagen. At the same concentration, the cell adhesion activities of 3A5D29N, 3A5D2NT, and 3A5D1NT were significantly superior to those of the larger recombinant type III collagen. At the same concentration, the cell adhesion activities of 17S1NNT, 17S3NT, and 17S28 were not significantly different from those of the larger recombinant type XVII collagen. This shows that although recombinant small molecule collagen has fewer amino acid residues and shorter sequences, their amino acid sequence design achieves a balance between small molecular weight (i.e. short sequence) and maintaining biological activity (i.e. maintaining a certain biological active site sequence).

[0302] (2) Cell proliferation detection

[0303] The cell proliferation activity detection method of recombinant small molecule collagen refers to the "Three General Rules 3528 Human Epidermal Growth Factor Biological Activity Assay" in the 2020 edition of the "Pharmacopoeia of the People's Republic of China".

[0304] Brief description of specific implementation methods:

[0305] Lyophilized recombinant small molecule collagen 3A5D29N and 17S28 were dissolved in pure water, the pH was adjusted to 7.2-7.4, sterilized by filtration through a 0.22 μm filter membrane, and diluted to 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL with sterile maintenance culture medium (0.5% FBS + 95.5% DMEM(H)). hEGF (purchased from Lonza) was used as a positive control at concentrations of 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL, and 0.125 ng / mL, and maintenance culture medium was used as a negative control. HaCat cell line (purchased from the Chinese Academy of Sciences Cell Bank, catalog number SCSP-5091) was cultured in complete culture medium (88% DMEM + 10% FBS + 1% glutamine + 1% sodium pyruvate) at 37°C and 5% carbon dioxide, and the cell concentration was controlled to be 1.0×10 cells per mL. 5 ~5.0×10 5 The cells were used for biological activity assay 24 to 36 hours after passage. The culture medium in the culture flask was discarded, the cells were digested and collected, and the complete culture medium was used to prepare a mixture containing 5.0×10 4A cell suspension of 100 cells was plated in a 96-well cell culture plate, 100 μL per well, and incubated at 37°C, 5% CO2. After 24 hours, the suspension was replaced with maintenance medium and incubated at 37°C, 5% CO2 for 24 hours. The maintenance medium was discarded from the prepared cell culture plate, and 100 μL of the control sample solution and the test sample solution were added to each well. The plates were incubated at 37°C, 5% CO2 for 24-72 hours. The 96-well plate was removed, and cell morphology was observed under a microscope. The liquid was then removed, and 50 μL of MTT solution (purchased from Shanghai Biyuntian Biotechnology Co., Ltd., prepared in DPBS, at a concentration of 1 mg / mL) was added to each well. The plates were then incubated at 37°C, 5% CO2. After 4 hours, the supernatant was removed, and 100 μL of isopropanol was added to each well to dissolve the crystals. The absorbance was measured at 570 nm using a microplate reader with a reference wavelength of 650 nm, and the results were recorded.

[0306] As shown in Figure 27, hEGF, used as a positive control, promoted the proliferation of HaCat cells. Recombinant small molecule collagens 3A5D29N and 17S28 also promoted the proliferation of HaCat cells at concentrations of 0.125 to 1 mg / mL. In particular, the proliferation activity of 17S28 in promoting HaCat cells showed an increasing trend with increasing sample concentration. The proliferation-promoting effect of higher concentrations of 17S28 was basically equivalent to that of 0.5 ng / mL hEGF.

[0307] (3) Transdermal absorption test

[0308] Using a full-thickness skin model as a carrier, the test sample was fluorescently labeled with FITC and evenly applied to the model surface using a topical administration method. The skin penetration behavior of the sample was observed by calculating the fluorescence intensity of the fluorescent slices and measuring the receiving solution using a fluorescence microplate reader to calculate the diffusion percentage. The testing was conducted by Shaanxi Boxi General Testing Technology Co., Ltd.

[0309] Brief description of specific implementation methods:

[0310] Recombinant small molecule collagen 3A5D29N, recombinant type III collagen (Jiangsu Chuangjian Medical Technology Co., Ltd., patent application number CN201310033299.6, published on May 15, 2013, molecular weight 43.6KDa), hydrolyzed hyaluronic acid (molecular weight ≤ 5000Da), and fish skin collagen (Nitta Gelatin Inc. Maringen SP03 (PF)) were labeled with FITC (fluorescein isothiocyanate) and purified with molecular sieves to remove uncoupled FITC. (Guangdong Boxi Biotechnology Co., Ltd.) The tissue was transferred to a 6-well plate, and 2 mL of 3D full-thickness skin model culture medium was added to each well.

[0311] According to the test group, samples of different concentrations were prepared. The sample group was administered surfaceally with a volume of 20 μL. When the culture reached the appropriate time, the residual test substance on the surface of the model could be cleaned with a sterile PBS solution washing bottle, and the residual liquid inside and outside the model was gently wiped with a sterile cotton swab. After the model was circumcised, it was immersed in a 4% paraformaldehyde solution for fixation (fixation time ≥ 24 h). After freezing and sectioning, fluorescence photography was performed. The IPP software was used to perform data statistics on the cumulative optical density (IOD) of the target substance in the image. The t-test statistical analysis (two-tailed) was used for inter-group comparison. The culture medium was collected and the fluorescence IOD value was detected. According to the fluorescence standard curve of different samples, the amount of sample that penetrated into the culture medium at 12 h was calculated. The diffusion percentage = sample amount / theoretical content of the sample × 100%, that is, the cumulative transdermal rate.

[0312] As shown in Figure 28, at 1 hour, fluorescence signals were detected in the full-thickness skin model for 1 mg / mL recombinant small-molecule collagen 3A5D29N and recombinant type III collagen, while no fluorescence signals were detected for the same concentrations of hydrolyzed hyaluronic acid and fish skin collagen. At this point, the FTIC-labeled recombinant small-molecule collagen sample had penetrated the epidermis and entered the skin, but had not yet fully penetrated the full-thickness skin model tissue. Fluorescence signal intensity analysis showed that the fluorescence signal of recombinant small-molecule collagen 3A5D29N was significantly stronger than that of the other three samples, indicating that its transdermal rate was significantly faster than that of recombinant type III collagen, hydrolyzed hyaluronic acid, and fish skin collagen. Furthermore, as shown in Figure 29, at 12 hours, the recombinant small-molecule collagen sample had completely penetrated the full-thickness skin model tissue. With increasing dosing concentration, the diffusion percentage (i.e., cumulative transdermal rate) of recombinant small-molecule collagen 3A5D29N continued to increase. At 1 mg / mL, it was essentially the same as the diffusion percentage (i.e., cumulative transdermal rate) of hydrolyzed hyaluronic acid at the same concentration, both exceeding 40%. These results demonstrate that recombinant small-molecule collagen can achieve rapid skin penetration in a short period of time and maintain a high cumulative penetration rate over an extended period. The full-thickness skin model tissue test results in this example demonstrate the excellent skin permeability of the small-molecule collagen of the present invention. This also suggests that recombinant small-molecule collagen has a promising future in the development of related products, such as cosmetics.

Claims

1. A recombinant small molecule collagen expression system, It is characterized in that The expression system includes a chassis cell or chassis engineering bacteria obtained by transforming a host bacterium with a localized fusion functional protein connection vector and a tandemly repeated expression sequence of a recombinant small molecule collagen protein; The tandem repeat expression sequence is repeated in series with recombinant small molecule collagen, artificially designed collagen with a typical GXY triplet structure, or collagen composed of two or more regions of human collagen sequence as the basic unit; there are recognition and cleavage sites for Kex2 enzyme and CPB enzyme between each two adjacent basic units in the tandem repeat expression sequence; The localization fusion functional protein includes a CPB enzyme and a functional region with intracellular membrane localization or conversion and transport between various organelles; the localization fusion functional protein also includes a connection sequence, which is used for connection between the CPB enzyme and the functional region sequence with intracellular membrane localization or conversion and transport between various organelles when they are fused and expressed; The localization fusion functional protein is an amino acid sequence as shown in SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26 or SEQ ID NO.28; The chassis cells or chassis engineering bacteria are selected from yeast.

2. The expression system according to claim 1, It is characterized in that The tandem repeat expression sequence may further include a recognition and cleavage site for the Ste13 enzyme between each two adjacent basic units.

3. The expression system according to claim 2, It is characterized in that The sites recognized and cleaved by the Kex2 enzyme include KR or RR dibasic amino acid residues, followed by EA, EAEA or other amino acid residues that facilitate recognition and cleavage by the Kex2 enzyme or Ste 13 enzyme.

4. The expression system according to claim 1, It is characterized in that The sites recognized and cut by the CPB enzyme include the basic amino acid residues at the carboxyl terminal of the protein, including K and R.

5. The expression system according to any one of claims 1 to 4, It is characterized in that The recombinant small molecule collagen has an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.63 or SEQ ID NO.

67.

6. The expression system according to claim 5, It is characterized in that The carboxyl terminus of the recombinant small molecule collagen has multiple His.

7. The expression system according to claim 6, It is characterized in that The recombinant small molecule collagen has an amino acid sequence as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.

8.

8. The expression system according to claim 1, It is characterized in that The tandemly repeated expression sequence is a sequence shown in SEQ ID NO.30, SEQ ID NO.33, SEQ ID NO.36, SEQ ID NO.39, SEQ ID NO.42, SEQ ID NO.45, SEQ ID NO.48, SEQ ID NO.51, SEQ ID NO.54, SEQ ID NO.57, SEQ ID NO.60, SEQ ID NO.64 or SEQ ID NO.

68.

9. The expression system according to claim 8, It is characterized in that The nucleic acid encoding the tandemly repeated expression sequence is a sequence shown in SEQ ID NO.71-81, or a degenerate sequence thereof.

10. The expression system according to claim 1, It is characterized in that The CPB enzyme is derived from human or rat.

11. The expression system according to claim 10, It is characterized in that The CPB enzyme sequence is shown in SEQ ID NO.17-18.

12. The expression system according to claim 1, It is characterized in that The functional region sequence with intracellular membrane localization or conversion and transport between organelles is derived from Kex2 enzyme of Saccharomyces cerevisiae or Pichia pastoris.

13. The expression system according to claim 12, It is characterized in that The functional region sequences having intracellular membrane localization or conversion and transport functions between organelles are shown in SEQ ID NOs. 19-20.

14. The expression system according to claim 1, It is characterized in that The connecting sequence is a Linker sequence, as shown in SEQ ID NO.21: GGSGSGSGGS.

15. The expression system according to claim 1, It is characterized in that The nucleic acid encoding the localization fusion functional protein is a nucleotide sequence shown in SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27 or SEQ ID NO.29, or a degenerate sequence thereof.

16. The expression system according to claim 1, It is characterized in that The chassis cells or chassis engineering bacteria are deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, and the deposit numbers are: CGMCC No.25815, CGMCC No.25817, CGMCC No.25816, and CGMCC No.25818.

17. Use of the expression system according to any one of claims 1 to 16 in obtaining recombinant small molecule collagen.

18. A method for preparing recombinant small molecule collagen, It is characterized in that The method adopts the expression system according to any one of claims 1 to 16, and the method comprises: Constructing a tandem repeat expression sequence of a recombinant small molecule collagen; the tandem repeat expression sequence is tandemly repeated with recombinant small molecule collagen, or artificially designed collagen with a typical GXY triplet structure, or a collagen composed of two or more regions of a human collagen sequence as a basic unit; there are recognition and cleavage sites for Kex2 enzyme and CPB enzyme between each two adjacent basic units in the tandem repeat expression sequence; Constructing a localization fusion functional protein, connecting the localization fusion functional protein to a vector and transforming the host bacteria to obtain chassis cells or chassis engineering bacteria; The tandem repeat expression sequence of the recombinant small molecule collagen is connected to an expression vector and then transferred into chassis cells or chassis engineering bacteria to obtain recombinant engineering bacteria containing or expressing the recombinant small molecule collagen, and the expression is induced by fermentation to obtain the recombinant small molecule collagen.

19. The method according to claim 18, It is characterized in that The recombinant engineered bacteria containing or expressing recombinant small molecule collagen are deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, and the deposit numbers are: CGMCC No.25819, CGMCC No.25821, CGMCC No.25827, CGMCC No.25829, CGMCC No.25828, CGMCC No.25820, and CGMCC No.25822.

20. The method according to claim 18, It is characterized in that The recombinant small molecule collagen obtained by the method has an amino acid sequence shown by SEQ ID NO.32, SEQ ID NO.35, SEQ ID NO.38, SEQ ID NO.41, SEQ ID NO.44, SEQ ID NO.47, SEQ ID NO.50, SEQ ID NO.53, SEQ ID NO.56, SEQ ID NO.59, SEQ ID NO.62, SEQ ID NO.66 or SEQ ID NO.

70.

21. The recombinant small molecule collagen obtained according to the method according to any one of claims 18 to 20.

22. Use of the recombinant small molecule collagen obtained by the method according to any one of claims 18 to 20, or the recombinant small molecule collagen according to claim 21 in the preparation of biomaterials, tissue engineering products, and cosmetics.