Self-assembling collagen molecules, recombinant human type III analog collagen, polynucleotides, recombinant expression vectors, recombinant host cells, and methods for preparing recombinant human type III analog collagen.

Abstract

The invention proposes a collagen polymer biomaterial made to resemble recombinant human type III self-crosslinking and a method for preparing such material. The collagen self-assembly element comprises (i) at least 21 consecutive amino acids at the C-terminus of the amino acid sequence as shown in Seq ID No. 1; or an amino acid sequence which is obtained by replacing, deleting, or adding one or more amino acids in the amino acid sequence as described in (i) and which retains the function of promoting the crosslinking of recombinant human type III self-crosslinking to produce a triple helix structure; or an amino acid sequence which has at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence as described in (i) and which retains the function of promoting the crosslinking of recombinant human type III self-crosslinking to produce a triple helix structure.

Description

Self-crosslinking recombinant humanized collagen polymer biomaterial and preparation method thereof

[0001] Citation of Related Applications

[0002] This disclosure claims priority to an invention patent application filed with the Patent Office of China on August 4, 2023, with application number 202310983181.3 and title “Self-crosslinked recombinant humanized collagen polymer biomaterial and its preparation method,” and the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure belongs to the field of synthetic biology, and specifically relates to a self-crosslinking recombinant humanized collagen polymer biomaterial and a preparation method thereof; more specifically, it relates to a collagen self-assembly element that promotes the self-crosslinking of recombinant humanized type III collagen to form a triple helical structure, and a corresponding recombinant humanized type III collagen with a triple helical structure. Background Art

[0004] Collagen is a protein widely distributed throughout human connective tissue and is the most abundant protein in the human body, accounting for 25% to 35% of the total protein content. Collagen's primary functions include maintaining the extracellular environment, upholding the normal physiological functions of tissues and organs, and repairing body damage. Collagen is a natural biological resource, possessing unmatched biocompatibility, cell-supporting elasticity, and biodegradability. Therefore, collagen is widely used in industries such as medicine and cosmetics.

[0005] Natural collagen molecules form a unique superhelical structure, a left-handed helix based on a basic repeating structure of three amino acid residues, typically in the form of Gly-X-Pro. Gly is essential for hydrogen bonding within collagen and lacks side chains, thus enabling dense packing of collagen. At higher levels of structure, collagen superhelices further associate to form collagen fibrils. In vivo, collagen synthesis and modification, starting with tropocollagen, undergo numerous chemical changes, including hydroxylation, glycosylation, and cross-linking, and are intricately regulated by a variety of enzymes. Tropocollagen contains not only collagen chains but also globular head and tail segments. Without these segments, the collagen chains fail to fold into the correct triple helix, resulting in a lack of biological activity. Consequently, collagen produced according to the original gene sequence struggles to spontaneously organize into the correct spatial structure in vitro. This difficulty has severely hampered the development and production of human collagen.

[0006] Appropriate research has been conducted on this issue. For example, Reference 1 discloses that the addition of hinge region amino acids such as GPPGPCCGGG (SEQ ID NO. 15) to the C-terminus of an expressed polypeptide can aid gel formation. However, Reference 2 discloses that the addition of hinge region amino acids such as those shown in SEQ ID NO. 15 to the C-terminus of a provided polypeptide sequence causes the polypeptide to form a gel-like precipitate during fermentation, making it difficult to dissolve and purify, and significantly reducing the yield.

[0007] Therefore, there is an urgent need for a method that can promote the cross-linking of recombinant humanized type III collagen to form a triple helical structure, so that recombinant humanized type III collagen can be mass-produced through biosynthesis and can have a triple helical structure and exert its specific functions.

[0008] Circular dichroism (CD) spectroscopy is a method used to examine protein secondary structure. When examining native collagen, an absorption peak around 221 nm appears under appropriate detection conditions. This absorption peak is related to its triple helical structure but is also influenced by factors such as the proline and hydroxyproline content (polyproline itself exhibits a strong peak at 221 nm, but does not form a triple helix). Therefore, CD spectroscopy cannot serve as direct evidence of a triple helical structure. However, due to its simplicity, CD spectroscopy can be used to assist in characterizing collagen's higher-order structure. CD spectroscopy testing conditions and results vary significantly for different collagens. This is primarily due to differences in sequence length and amino acid composition (especially proline and hydroxyproline content) between recombinant collagen and native full-length collagen. Consequently, CD spectroscopy testing conditions (temperature, concentration, solution formulation, etc.) cannot be consistent across different collagens. To determine whether collagen can form a triple helix (for example, if collagen denatured during production or defectively designed is completely unable to form a triple helix), CD spectroscopy can be used to characterize collagen under different conditions.

[0009] References:

[0010] Cited literature 1: Yao J, Yanagisawa S, Asakura T. Design, expression and characterization of collagen-like proteins based on the cell adhesive and crosslinking sequences derived from native collagens. J Biochem. 2004 Nov; 136(5):643-9.

[0011] Reference 2: CN 109593126 B.

[0012] Summary of the Invention

[0013] Problems to be solved by the invention

[0014] At present, the existing technology has conducted appropriate research on the problem that, for example, collagen prepared according to the original gene sequence is difficult to spontaneously organize into the correct spatial structure in vitro, resulting in its inability to effectively perform physiological functions, but it still cannot be said to be sufficient. In this regard, the present disclosure provides a collagen self-assembly element, which can promote the self-crosslinking of recombinant type III humanized collagen to form a triple helical structure, and by making the end of the amino acid sequence of the recombinant type III humanized collagen contain the amino acid sequence of the hinge region of human type III collagen, the recombinant type III humanized collagen is helped to self-crosslink to form a triple helical structure, and the recombinant type III humanized collagen biomaterial with a triple helical structure can be successfully prepared by a biosynthetic method.

[0015] Solutions for solving problems

[0016] In a first aspect, the present disclosure provides a collagen self-assembly element, wherein the collagen self-assembly element comprises a sequence as shown in any one of the following (i) to (iii):

[0017] (i) at least 21 consecutive amino acids at the C-terminus of the amino acid sequence shown in SEQ ID NO. 1;

[0018] (ii) an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence described in (i), while retaining the function of promoting the self-crosslinking of recombinant humanized type III collagen to form a triple helical structure;

[0019] (iii) an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described in (i) and retains the function of promoting the self-crosslinking of recombinant humanized type III collagen to form a triple helical structure.

[0020] In some embodiments, the sequence shown in (i) is an amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0021] The second aspect of the present disclosure provides use of the collagen self-assembly element according to the first aspect of the present disclosure in promoting self-crosslinking of recombinant humanized type III collagen to form a triple helical structure.

[0022] In some embodiments, the collagen self-assembly element functions by being present at the end of the amino acid sequence of the recombinant humanized type III collagen;

[0023] Preferably, the end of the amino acid sequence of the recombinant humanized type III collagen is the C-terminus.

[0024] A third aspect of the present disclosure provides a recombinant humanized type III collagen, wherein the end of the recombinant humanized type III collagen comprises the collagen self-assembly element according to claim 1 or 2;

[0025] Preferably, the terminus of the recombinant humanized type III collagen is the C-terminus.

[0026] In some embodiments, the recombinant humanized type III collagen comprises the sequence shown in any one of the following (a) to (c):

[0027] (a) the amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9;

[0028] (b) an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence as described in (i), and the amino acid sequence retains the ability to self-crosslink to form a triple helical structure;

[0029] (c) an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described in (i) and retains the ability to self-crosslink to form a triple helical structure.

[0030] The fourth aspect of the present disclosure provides a polynucleotide encoding the collagen self-assembly element according to the first aspect of the present disclosure, or encoding the recombinant humanized type III collagen according to the third aspect of the present disclosure.

[0031] In some embodiments, the polynucleotide comprises the sequence shown in (z1) or (z2) below:

[0032] (z1) the nucleotide sequence shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14;

[0033] (z2) A nucleotide sequence that hybridizes with the nucleotide sequence shown in (z1) under stringent conditions and encodes a protein having the ability to self-crosslink to form a triple helical structure, wherein the stringent conditions are moderately stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

[0034] The fifth aspect of the present disclosure provides a recombinant expression vector comprising the polynucleotide according to the fourth aspect of the present disclosure.

[0035] The sixth aspect of the present disclosure provides a recombinant host cell, which comprises the recombinant expression vector according to the fifth aspect of the present disclosure.

[0036] A seventh aspect of the present disclosure provides a method for preparing the recombinant humanized type III collagen according to the third aspect of the present disclosure, the method comprising the following steps:

[0037] S1: constructing a recombinant expression vector comprising the polynucleotide encoding the recombinant humanized type III collagen according to the third aspect according to the fourth aspect, and constructing a recombinant host cell by transformation;

[0038] S2: culturing the recombinant host cell obtained in S1 in a culture medium and producing a protein;

[0039] S3: harvesting and purifying the protein, preferably purifying the protein using a Ni column and / or anion exchange chromatography; and

[0040] S4: Optionally, the protein is cleaved by enzyme, preferably by using TEV protease.

[0041] Effects of the Invention

[0042] Through the implementation of the above technical solutions, the present disclosure achieves the following technical effects:

[0043] First, the present disclosure provides a collagen self-assembly element. By making the end of the amino acid sequence of recombinant humanized type III collagen contain the collagen self-assembly element, that is, containing the amino acid sequence of the hinge region of human type III collagen, it can effectively help the recombinant humanized type III collagen to self-crosslink to form a triple helix structure, thereby greatly improving the availability of recombinant humanized type III collagen prepared by biosynthesis methods.

[0044] Furthermore, the recombinant humanized type III collagen provided herein, which contains a collagen self-assembly element at its terminus, specifically the amino acid sequence of the hinge region of human type III collagen, can form a well-defined triple-helical structure, a finding supported by circular dichroism spectroscopy. Furthermore, the recombinant humanized type III collagen can be produced in large quantities through biosynthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 shows the electrophoresis detection results during the preparation of recombinant type III humanized collagen TE16c.

[0046] FIG2 shows the electrophoresis detection results during the preparation of recombinant humanized type III collagen C3T16H1.

[0047] FIG3 shows the electrophoresis detection results during the preparation of recombinant humanized type III collagen C3T16H2.

[0048] FIG4 shows the electrophoresis detection results during the preparation of recombinant humanized type III collagen C3T16H3.

[0049] FIG5 shows the electrophoresis detection results during the preparation of recombinant humanized type III collagen C3T16H4.

[0050] Figure 6 shows the circular dichroism UV scanning analysis results of recombinant type III humanized collagen C3T16H1, C3T16H2, C3T16H3, C3T16H4 and TE16c, and a positive peak near 221 nm was detected. DETAILED DESCRIPTION

[0051] The following describes the embodiments of the present disclosure, but the present disclosure is not limited thereto. The present disclosure is not limited to the various structures described below, and various modifications can be made within the scope of the disclosure. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present disclosure.

[0052] In the present disclosure, a numerical range expressed using "a value A to a value B" or "a value A - a value B" means a range including the endpoints A and B.

[0053] In the present disclosure, a numerical range expressed using "above" or "below" means a numerical range including the number.

[0054] In this disclosure, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0055] In the present disclosure, the term “a” or “an” or “the” may mean “one”, and may also mean “one or more”, “at least one” and “one or more than one”.

[0056] In this disclosure, the terms "comprising," "having," "including," or "containing" may be inclusive or open-ended, and do not exclude additional, unrecited elements or method steps. At the same time, "comprising," "having," "including," or "containing" may also be closed-ended, excluding additional, unrecited elements or method steps.

[0057] In this disclosure, the term "about" may mean that a value includes the standard deviation of the error of the device or method used to determine the value. The numerical ranges and parameters used to define the present disclosure are all approximate values, and the relevant numerical values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations caused by the aforementioned testing devices or methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within ±10%, ±5%, ±3%, ±1% or ±0.5% of a particular value or range.

[0058] In this disclosure, the terms "polypeptide" and "protein" interchangeably refer to a string of at least two amino acid residues linked to each other by covalent bonds (e.g., peptide bonds), and can be a recombinant polypeptide, a naturally occurring polypeptide, or a synthetic polypeptide. A polypeptide can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0059] In this disclosure, the term "amino acid" may include natural amino acids, unnatural amino acids, amino acid analogs, and all their D and L stereoisomers.

[0060] In the present disclosure, amino acid modifications may include modifications to the native sequence, such as modification of functional groups, intramolecular covalent bonding (e.g., cyclization between side chains), methylation, acylation, ubiquitination, phosphorylation, aminohexanylation, biotinylation, etc.

[0061] In the present disclosure, amino acid deletion may refer to the deletion of 1, 2 or 3 or more amino acids from an amino acid sequence, as long as the altered sequence fully or partially retains the activity of the original amino acid sequence.

[0062] In the present disclosure, amino acid addition may refer to the addition of 1, 2 or 3 or more amino acids at the C-terminus, N-terminus or any position between the C-terminus and the N-terminus of an amino acid sequence, as long as the altered sequence fully or partially retains the activity of the original amino acid sequence.

[0063] In the present disclosure, amino acid substitution may refer to the replacement of an amino acid at a certain position in an amino acid sequence by another amino acid, as long as the altered sequence retains the activity of the original amino acid sequence in whole or in part. Amino acid substitution may be a conservative amino acid substitution, which refers to the replacement of several amino acids by amino acids with similar or similar properties compared to the original amino acid sequence to form a peptide (conservative variant peptide). Exemplary, these conservative variant peptides can be produced based on the following amino acid replacements: replacement of Ala by Val, Leu or Ile, replacement of Arg by Lys, Gln, Asn or His, replacement of Asn by Gln, His, Lys or Arg, replacement of Asn by Glu or Asn, replacement of Cys by Ser or Ala, replacement of Gln by Asn or Glu, replacement of Glu by Asp or Gln, replacement of Gly by Ala, replacement of His by Asn, Lys, Gln or Arg, replacement of Leu, Met, Ala, Val, Phe or norleucine by Val, The amino acid substitutions may also be non-conservative amino acid substitutions.

[0064] In the present disclosure, the terms "sequence identity" and "percentage identity" refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotides or polypeptides and scoring the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides, and comparing it with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. A polynucleotide can differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). A polypeptide can differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotides or polypeptides. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0065] In this disclosure, the terms "moderately stringent conditions," "medium-high stringency conditions," "high stringency conditions," or "very high stringency conditions" describe conditions for nucleic acid hybridization and washing. Guidance for conducting hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in this document, and either method can be used. For example, specific hybridization conditions are as follows: (1) low stringency hybridization conditions are in 6× sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2×SSC, 0.1% SDS at at least 50°C (the wash temperature can be increased to 55°C for low stringency conditions); (2) moderate stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C, and preferably; (4) very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2×SSC, 1% SDS at 65°C.

[0066] In this disclosure, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may have any three-dimensional structure and may perform any function, known or unknown.

[0067] In the present disclosure, the term "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into protein; and iii) appropriate transcription and translation start and stop sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not important, and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, DNA from viruses such as lentiviruses, retroviruses, vaccinia, adenoviruses, fowlpox, baculoviruses, SV40, and pseudorabies.

[0068] In this disclosure, the term "recombinant host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformed bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary) and NSO cells.

[0069] In the present disclosure, the term "codon optimized" means that a nucleotide sequence encoding a polypeptide has been configured to contain codons preferred by the host cell or organism to improve gene expression in the host cell or organism and increase translation efficiency.

[0070] Unless otherwise defined, other technical and scientific terms used in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0071] The following is a more detailed description of the technical solutions provided by the present disclosure:

[0072] Collagen self-assembly elements

[0073] The present disclosure provides a collagen self-assembly element, which can effectively promote the self-crosslinking of recombinant type III humanized collagen to form a triple helical structure.

[0074] In some embodiments, the collagen self-assembly element comprises a sequence as shown in any one of the following (i) to (iii):

[0075] (i) at least 21 consecutive amino acids at the C-terminus of the amino acid sequence shown in SEQ ID NO. 1;

[0076] (ii) an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence described in (i), while retaining the function of promoting the self-crosslinking of recombinant humanized type III collagen to form a triple helical structure;

[0077] (iii) an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described in (i) and retains the function of promoting the self-crosslinking of recombinant humanized type III collagen to form a triple helical structure.

[0078] SEQ ID NO.1: GERGSEGSPGHPGQPGPPGPPGAPGPCCGG, which is a human type III collagen peptide segment.

[0079] The present disclosure unexpectedly discovered that collagen self-assembly elements of different lengths have different abilities to promote the self-crosslinking of recombinant humanized type III collagen to form a triple helical structure.

[0080] In some specific embodiments, the amino acid sequence of the collagen self-assembly element comprises a sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0081] In some more specific embodiments, the amino acid sequence of the collagen self-assembly element is a sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0082] SEQ ID NO. 2: GSEGSPGHPGQPGPPGPPGAPGPCCGG.

[0083] SEQ ID NO. 3: GSPGHPGQPGPPGPPGAPGPCCGG.

[0084] SEQ ID NO. 4: GHPGQPGPPGPPGAPGPCCGG.

[0085] In order to effectively exert the function of the collagen self-assembly element in promoting the self-crosslinking of recombinant type III humanized collagen to form a triple helical structure, in some embodiments, the collagen self-assembly element is located at the end of the recombinant type III humanized collagen, preferably the C-terminus.

[0086] The recombinant humanized type III collagen disclosed herein refers to a full-length or partial amino acid sequence fragment encoded by the type III human collagen gene prepared by DNA recombination technology, or a combination containing functional fragments of type III human collagen.

[0087] In some specific embodiments, the amino acid sequence of the recombinant type III humanized collagen contains a collagen self-assembly element at the end, which can be achieved by designing the amino acid sequence of the recombinant type III humanized collagen containing a collagen self-assembly element at the end and its gene sequence, and constructing an expression vector containing the gene sequence, which is transformed into a host cell, and then the host cell can be fermented and cultured, and the recombinant type III humanized collagen can be induced to express.

[0088] Recombinant humanized type III collagen

[0089] The present disclosure provides a recombinant type III humanized collagen protein, the end of which contains a collagen self-assembly element. As described above, the recombinant type III humanized collagen protein provided by the present disclosure contains the collagen self-assembly element, which can self-cross-link to form a triple helical structure after expression, and the protein can be prepared in large quantities by a biosynthetic method.

[0090] In order to better form a triple helix structure, the collagen self-assembly element is located at the C-terminus of the recombinant type III humanized collagen.

[0091] In some specific embodiments, the recombinant humanized type III collagen comprises the sequence shown in any one of the following (a) to (c):

[0092] (a) the amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9;

[0093] (b) an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence as described in (i), and the amino acid sequence retains the ability to self-crosslink to form a triple helical structure;

[0094] (c) an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described in (i) and retains the ability to self-crosslink to form a triple helical structure.

[0095] In some preferred embodiments, the amino acid sequence of the recombinant humanized type III collagen is a sequence as shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9.

[0096] Polynucleotide, recombinant expression vector, recombinant host cell

[0097] The present disclosure provides polynucleotides encoding the aforementioned collagen self-assembly elements and the aforementioned recombinant humanized type III collagen.

[0098] In some embodiments, the polynucleotide encoding the recombinant humanized type III collagen comprises the sequence shown in (z1) or (z2) below:

[0099] (z1) the nucleotide sequence shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14;

[0100] (z2) A nucleotide sequence that hybridizes with the nucleotide sequence shown in (z1) under stringent conditions and encodes a protein having the ability to self-crosslink to form a triple helical structure, wherein the stringent conditions are moderately stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

[0101] The present disclosure provides a recombinant expression vector comprising the above-mentioned polynucleotide.

[0102] The expression vectors selected in the present disclosure can be stably present and autonomously replicated in various prokaryotic or eukaryotic host cells, such as conventional plasmids (pET series), shuttle vector PNV 18.1, phage or viral vectors, etc. The polynucleotide sequences of the present disclosure are cloned into the vector through molecular biological manipulations such as enzyme digestion and ligation to construct a recombinant expression vector.

[0103] The present disclosure provides a recombinant host cell comprising the above-mentioned recombinant expression vector.

[0104] The host cells described in the present disclosure are selected from prokaryotic cells, yeast or eukaryotic cells, and can further be selected from Escherichia coli, Rhodococcus erythrorhizium, Bacillus subtilis and yeast. The recombinant expression vector described in the present disclosure is transformed into the host cells to obtain the corresponding genetically engineered bacteria.

[0105] Preparation method of recombinant humanized type III collagen

[0106] The present disclosure provides a method for preparing the above-mentioned recombinant humanized type III collagen, which comprises the following steps:

[0107] S1: constructing a recombinant expression vector comprising a polynucleotide encoding the recombinant humanized type III collagen, and constructing a recombinant host cell by transformation;

[0108] S2: culturing the recombinant host cell in a culture medium and producing a protein;

[0109] S3: harvesting and purifying the protein, preferably purifying the protein using a Ni column and / or anion exchange chromatography; and

[0110] S4: Optionally, the protein is cleaved by enzyme, preferably by using collagenase.

[0111] Example

[0112] The embodiments of the present disclosure will be described in detail below with reference to the examples. However, those skilled in the art will appreciate that the following examples are intended only to illustrate the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials and instruments used were commercially available conventional products.

[0113] Example 1: Construction and expression of recombinant humanized type III collagen

[0114] 1. The amino acid sequences of different lengths of the hinge region of human type III collagen were combined with the amino acid sequence of recombinant type III humanized collagen (TE16c, described in the prior patent ZL201811438582.6 of the applicant of this application) to obtain recombinant type III humanized collagen containing hinge regions of different lengths.

[0115] (1) Amino acid sequence

[0116] Amino acid sequence of recombinant type III humanized collagen TE16c: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGP NGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP(SEQ ID NO.5).

[0117] Amino acid sequence of recombinant type III humanized collagen C3T16H1: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGSEGSPGHPGQPGPPGPPGAPGPCCGG (SEQ ID NO.6).

[0118] Amino acid sequence of recombinant humanized collagen type III C3T16H2: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGSEGSPGHPGQPGPPGPPGAPGPCCGG (SEQ ID NO.7).

[0119] Amino acid sequence of recombinant humanized collagen type III C3T16H3: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGSPGHPGQPGPPGPPGAPGPCCGG (SEQ ID NO.8).

[0120] The amino acid sequence of recombinant humanized type III collagen C3T16H4 is: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPA GPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGHPGQPGPPGPPGAPGPCCGG(SEQ ID NO.9).

[0121] In the above sequence, the underlined portion is the amino acid sequence of different lengths in the hinge region of human type III collagen, that is, collagen self-assembly elements of different lengths.

[0122] (2) Nucleotide sequence

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] 2. The gene sequences of recombinant humanized type III collagen proteins C3T16H1, C3T16H2, C3T16H3, and C3T16H4 were synthesized. The nucleotide sequences encoding each of these proteins (with a collagenase cleavage site at the 5' end containing the amino acid sequence ENLYFQ (SEQ ID NO. 16) and the nucleotide sequence gaaaacctgtatttccag (SEQ ID NO. 17)) were inserted between the KpnI and XhoI restriction sites of the pET-28a-Trx-His expression vector. The TE16c recombinant expression plasmid is an existing plasmid described in the applicant's prior patent ZL201811438582.6.

[0129] 3. Transform the successfully constructed expression plasmid into E. coli competent cells BL21 (DE3). The specific process is as follows: (1) Take out the E. coli competent cells BL21 (DE3) from the ultra-low temperature refrigerator and place them on ice. When they are half melted, take 2 μl of the plasmid to be transformed and add it to the E. coli competent cells BL21 (DE3) and mix it slightly 2-3 times. (2) Place the mixture on ice for 30 minutes, then heat shock it in a 42°C water bath for 45-90 seconds, take it out and place it on ice for 2 minutes. (3) Transfer it to a biosafety cabinet and add 700 μl of liquid LB culture medium, then incubate it at 37°C and 220 rpm for 60 minutes. (4) Take 200 μl of the bacterial solution and evenly spread it on an LB plate containing ampicillin sodium. (5) Incubate the plate in a 37°C incubator for 15-17 hours until colonies of uniform size grow.

[0130] 4. Pick 5-6 single colonies from the transformed LB plates and place them in a shake flask containing antibiotic stock solution (single colonies transformed with the C3T16H1, C3T16H2, C3T16H3, and C3T16H4 recombinant expression plasmids should be added to an antibiotic stock solution containing kanamycin sulfate; single colonies transformed with the TE16c recombinant expression plasmid should be added to an antibiotic stock solution containing ampicillin sodium). Incubate in a shaker at 220 rpm and 37°C for 7 hours. Cool the shake flask to 16°C and add IPTG to induce expression for a period of time. Then, aliquot the culture solution into centrifuge bottles and centrifuge at 8000 rpm and 4°C for 10 minutes. Collect the cells, record their weight, and take samples for electrophoresis.

[0131] 5. Resuspend the collected cells in balanced working solution (200mM NaCl, 25mM Tris, 20mM imidazole, pH 8.0). Cool the culture solution to ≤15°C and homogenize. High-pressure homogenize twice (mark the homogenized sample as "homogenized"). After completion, collect the culture solution. Aliquot the homogenized culture solution into centrifuge bottles and centrifuge at 17,000 rpm and 4°C for 30 minutes. Collect the supernatant and take the supernatant (marked as "supernatant") and the precipitate (marked as "precipitate") for electrophoresis.

[0132] 6. Purify and digest the collected supernatant. The specific process is as follows: (1) Crude purification: a. Wash the column (Ni6FF, Cytiva) with water for 5 CV. b. Equilibrate the column with equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) for 5 CV. c. Loading: Add the collected supernatant to the column until the liquid flows out, and take the flow-through for electrophoresis (marked: flow-through). d. Wash impurities: Add 25 mL of wash solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) until the liquid flows out, and take the wash flow-through for electrophoresis (marked: wash impurities). e. Collect the target protein: add 20 mL of eluent (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0) and collect the flow-through (labeled: elution). Detect the protein concentration by UV-visible spectrophotometry and perform electrophoresis. f. Wash the column with 1 M imidazole working solution (labeled: 1 M wash). g. Wash the column with purified water. (2) Enzymatic digestion: Add collagenase at a protein to collagenase mass ratio of 20:1, digest at 16°C for 4 h, and take a sample (labeled: enzyme digestion) for electrophoresis. Place the protein solution after enzymatic digestion in a dialysis bag with dialysis solution A (20 mM Tris, 20 mM sodium chloride, pH 8.0). Dialyze at 4°C for 2 h, then transfer to new dialysis solution for overnight dialysis at 4°C. Take a sample (labeled: change solution) for electrophoresis. (3) Purification: a. Equilibrate the column (Capto Q, Cytiva): Equilibrate the column with Solution A (20 mM Tris, 20 mM NaCl, pH 8.0) at a flow rate of 10 ml / min. b. Load the sample: Load the sample at a flow rate of 5 ml / min and collect the flow-through (labeled as Q flow-through) for electrophoresis. c. Binary mobile phase gradient elution: Set the mobile phases to 0% (V / V)-15% (V / V) solution B (20 mM Tris, 1 M NaCl, pH 8.0) and 100% (V / V)-85% (V / V) solution A for 2 min, then hold for 3 CVs; then 15% (V / V)-30% (V / V) solution B and 85% (V / V)-70% (V / V) solution A for 2 min, then hold for 3 CVs; then 30% (V / V)-50% (V / V) solution B and 70% (V / V)-50% (V / V) solution A for 2 min, then hold for 3 CVs; then 50% (V / V)-100% (V / V) solution B and 50% (V / V)-0% (V / V) solution A for 2 min, then hold for 3 CVs. Collect peaks (labeled: B wash) and perform electrophoresis. d. Clean the column. e. Detect the target protein content and calculate the protein yield, and store the protein at 4°C.

[0133] 7. Concentration detection

[0134] (1) Crude protein concentration detection

[0135] Accurately measure an appropriate amount of sample, dilute 10-50 times with eluent, and stir thoroughly with a glass rod. Measure the absorbance at 280 nm using a UV-visible spectrophotometer. Calculate the protein concentration using the formula C (mg / ml) = A280 × absorbance coefficient × dilution factor (Note: The absorbance value must be between 0.1 and 1).

[0136] The concentration test results are as follows:

[0137] The protein expression levels were C3T16H3>C3T16H4>C3T16H1>C3T16H2>TE16c.

[0138] (2) Detection of purified protein concentration

[0139] Accurately measure an appropriate amount of sample, dilute 10-50 times with eluent, and stir thoroughly with a glass rod. Measure absorbance at 215 and 225 nm using a UV-Vis spectrophotometer. Calculate protein concentration using the formula C (mg / ml) = (A215 - A225) × 144 × dilution factor ÷ 1000 (Note: absorbance must be between 0.1 and 1).

[0140] The concentration test results are as follows:

[0141] The protein content after purification is C3T16H4>C3T16H1>C3T16H2>C3T16H3>TE16c.

[0142] 8. Electrophoresis detection

[0143] The specific process is as follows: 40 μl of sample solution was taken, 10 μl of 5× protein loading buffer (250 mM Tris-HCl (pH 6.8), 10% (V / V) SDS, 0.5% (V / V) bromophenol blue, 50% (V / V) glycerol, 5% (V / V) β-mercaptoethanol) was added, and the sample was boiled in 100°C water for 10 min. Then, 10 μl per well was added to SDS-PAGE protein gel, and electrophoresis was performed at 80 V for 2 h. The protein was stained with Coomassie brilliant blue staining solution (0.1% (V / V) Coomassie brilliant blue R-250, 25% (V / V) isopropanol, 10% (V / V) glacial acetic acid) for 20 min, and then decolorized with protein destaining solution (10% (V / V) acetic acid, 5% (V / V) ethanol).

[0144] The electrophoresis detection results are shown in Figures 1 to 5. The expression levels of C3T16H1, C3T16H2, C3T16H3, C3T16H4, and TE16c proteins were all good, and the purity of the purified collagen was good without obvious miscellaneous bands.

[0145] Example 2: UV-scanning analysis of recombinant humanized type III collagen by circular dichroism

[0146] Experimental methods

[0147] (1) Instrument parameter setting

[0148] Band width: 1.0nm;

[0149] Step: 1.0nm;

[0150] Measurement range: 190-260nm (far-UV region scan) / 250-340nm (near-UV region scan);

[0151] Time per point: 0.5s;

[0152] Repeats: 3 times;

[0153] Cuvette length (Cell Length): 10mm|0.5mm;

[0154] Temperature: Room Temperature.

[0155] (2) Far and near UV scanning of standard products

[0156] The scanning wavelength was set to 180-340 nm for background test and blank buffer test, and then the far- and near-UV circular dichroism absorption of 1 mg / mL CSA standard solution in the range of 180-340 nm was collected.

[0157] (3) Sample processing

[0158] Purified C3T16H1, C3T16H2, C3T16H3, C3T16H4, and TE16c protein samples were collected and concentrated to a protein concentration of 1 mg / ml using 10 kDa ultrafiltration concentrators (Millipore).

[0159] (4) Sample far-UV scanning

[0160] Soak the cuvette in 2M HNO3 overnight, rinse with deionized water and dry, collect the background first, then collect the blank buffer, then add an appropriate amount of test sample to the cuvette, perform far-UV scanning at 190-260nm according to the above parameters and collect data.

[0161] (5) Sample near-UV scanning

[0162] Soak the cuvette in 2M HNO3 overnight, rinse with deionized water and dry it. Collect the background first, then collect the blank buffer solution, then add an appropriate amount of test sample to the cuvette, perform near-UV scanning at 250-340nm according to the above parameters and collect data.

[0163] (6) Scanning spectrum processing

[0164] All scanned images were processed by subtracting baseline and smoothing using the software Pro-Data Viewer.

[0165] Experimental results and analysis

[0166] The experimental results are shown in Figure 6. C3T16H1, C3T16H2, C3T16H3, C3T16H4, and TE16c all have positive peaks at 221 nm, and the positive peak values ​​are C3T16H2>C3T16H4≥C3T16H3>C3T16H1≥TE16c. This indicates that C3T16H1, C3T16H2, C3T16H3, C3T16H4, and TE16c all have triple-helical structures at the same protein concentration. The amino acid sequence of the hinge region of human type III collagen contributes to the formation of the triple-helical structure of recombinant humanized type III collagen, and the amino acid sequences of hinge regions of different lengths have different abilities to promote the formation of the triple-helical structure of recombinant humanized type III collagen.

Claims

1. A collagen self-assembly element, characterized in that: The collagen self-assembly element comprises a sequence as shown in any one of the following (i) to (iii): (i) at least 21 consecutive amino acids at the C-terminus of the amino acid sequence shown in SEQ ID NO.1; (ii) an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence described in (i), and the amino acid sequence retains the function of promoting the self-crosslinking of recombinant humanized type III collagen to form a triple helical structure; (iii) an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described in (i), and retains the function of promoting the self-crosslinking of recombinant humanized type III collagen to form a triple helical structure.

2. The collagen self-assembly element according to claim 1, characterized in that: The sequence shown in (i) is an amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.

4.

3. Use of the collagen self-assembly element according to claim 1 or 2 in promoting the self-crosslinking of recombinant type III humanized collagen to form a triple helical structure.

4. The use according to claim 3, characterized in that The collagen self-assembly element plays a role by being present at the end of the amino acid sequence of the recombinant type III humanized collagen; Preferably, the end of the amino acid sequence of the recombinant humanized type III collagen is the C-terminus.

5. A recombinant humanized type III collagen, characterized in that: The end of the recombinant humanized type III collagen comprises the collagen self-assembly element according to claim 1 or 2; Preferably, the terminus of the recombinant humanized type III collagen is the C-terminus.

6. The recombinant humanized type III collagen according to claim 5, characterized in that: The recombinant humanized type III collagen comprises a sequence as shown in any one of the following (a) to (c): (a) the amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9; (b) an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence as described in (i), and the amino acid sequence retains the ability to self-crosslink to form a triple helical structure; (c) an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described in (i) and retains the ability to self-crosslink to form a triple helical structure.

7. A polynucleotide, characterized in that The polynucleotide encodes the collagen self-assembly element according to claim 1 or 2, or encodes the recombinant type III humanized collagen according to claim 5 or 6; Preferably, the polynucleotide comprises the sequence shown in (z1) or (z2): (z1) the nucleotide sequence shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14; (z2) A nucleotide sequence that hybridizes with the nucleotide sequence shown in (z1) under stringent conditions and encodes a protein having the ability to self-crosslink to form a triple helical structure, wherein the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

8. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the polynucleotide according to claim 7.

9. A recombinant host cell, characterized in that The recombinant host cell comprises the recombinant expression vector according to claim 8.

10. The method for preparing recombinant humanized type III collagen according to claim 5 or 6, characterized in that: The preparation method comprises the following steps: S1: constructing a recombinant expression vector comprising the polynucleotide encoding the recombinant humanized type III collagen according to claim 7, and constructing a recombinant host cell by transformation; S2: culturing the recombinant host cell obtained in S1 in a culture medium and producing a protein; S3: harvesting and purifying the protein, preferably purifying the protein using a Ni column and / or anion exchange chromatography; and S4: Optionally, the protein is cleaved by enzymes, preferably by using TEV protease.

Images