A recombinant human type III collagen and its expression system and application

Abstract

The present invention belongs to the field of bioengineering technology and relates to a recombinant human type III collagen protein, its expression system, and its application. The method for constructing the expression system comprises inserting the gene fragment of the recombinant human type III collagen protein into the expression plasmid of the expression host, transforming the expression host, and screening to obtain recombinant engineered bacteria with high expression. The amino acid sequence of the recombinant human type III collagen protein is shown in SEQ ID No. 2, and the nucleotide sequence of the encoding gene N3Aaa155-377 is shown in SEQ ID No. 3. The present invention also provides biomedical materials, cosmetics, or skin care products containing the recombinant human type III collagen protein.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering and relates to a recombinant human type III collagen and an expression system and application thereof. Background Art

[0002] Collagen is the most abundant and widely distributed protein in animals, accounting for approximately 25%-33% of the total protein in the body. It plays a vital role in maintaining the normal physiological function and repairing injuries of cells, tissues, and organs. Collagen is a rich variety of proteins, each with a diverse range of functions. Currently, approximately 29 types of collagen have been confirmed to exist in vertebrates, and they can be divided into two main categories based on their structural shape: fibrous and non-fibrous. Fibrous collagen primarily functions as a cellular scaffold, anchoring cells in place and providing tensile strength and stiffness to tissues. The main representative types are types I, II, III, V, and XI. Type III collagen is the predominant collagen type in young skin, providing support and moisture retention. It also interacts with other cells and proteins to maintain the normal physiological state of skin tissue. Type III collagen is composed of three single chains, which are hydrogen-bonded to form collagen's characteristic triple helical structure. The single chain is 1466 amino acids long, of which 1-23 are a signal peptide, 24-153 are an N-terminal propeptide, 154-1221 are the mature peptide chain, and 1222-1466 are the C-terminal propeptide. The mature peptide chain consists of an N-terminal domain, a triple helical region, and a C-terminal domain. The functional triple helical domain is formed by a "GXY" amino acid triplet, where G represents glycine, X represents any other amino acid, and Y is typically proline, which can be modified by a hydroxyl group to become hydroxylated proline within the cell. This formation of a triple helical structure is achieved through hydrogen bonding between the prolines of the three single chains. The triple helical region is the primary structural region for the function of type III collagen.

[0003] Type III collagen has a unique function and status in the skin, and is therefore favored in the development of skin-related biomedical materials and skin cosmetics. Since the full-length molecular weight of type III collagen is relatively large and its synthesis is extremely difficult, researchers usually intercept partial sequences of the triple helical region in type III collagen for full chemical synthesis or recombinant expression. When collagen itself is functioning, its molecular weight is larger than that of general polypeptides, so people tend to use collagen fragments with larger molecular weight (>10kD) for related research to be closer to the real situation of the human body. For collagen sequences with larger molecular weights, recombinant expression is a more common way to obtain them. However, since the collagen sequence has repeating units, its amino acid sequence is highly repetitive, which makes it more difficult to express, and is more prone to low expression levels or incorrect entanglement between different peptide chains during expression. Therefore, some researchers have chosen to express smaller fragments of the triple-helical region, typically a dozen or so amino acids, by repeatedly splicing them together to increase protein molecular weight and reduce the difficulty of expression. While this approach ensures fragment sequence homology, the resulting spliced ​​fragments do not actually exist in humans, resulting in insufficient overall sequence homology and conservation. On the other hand, some researchers have used eukaryotic expression systems, such as yeast and human cells, to express high-molecular-weight collagen fragments. While these systems offer greater expression capacity and a higher probability of correct protein structure, they face economic challenges such as limited culture density and high feed costs. To address these issues, Escherichia coli can be used as an expression host, with expression vector plasmids transferred into E. coli through genetic engineering. This expression system is one of the most commonly used in both laboratory and industrial production, offering strong reproduction capacity, high culture density, low feed costs, and high protein expression levels, making it economical and practical. Therefore, the design and development of a non-spliced, highly homologous, high-molecular-weight, and structurally intact recombinant human type III collagen fragment that can be expressed in a prokaryotic expression system holds considerable research value. Summary of the Invention

[0004] The purpose of the present invention is to provide a recombinant human type III collagen and its expression system and application.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides an expression system for recombinant human type III collagen, wherein the amino acid sequence of the recombinant human type III collagen is shown as SEQ ID No. 2.

[0007] The nucleotide sequence of the recombinant human type III collagen encoding gene N3Aaa155-377 is shown in SEQ ID No. 3.

[0008] Specifically, the method for constructing the expression system of recombinant human type III collagen provided by the present invention is to insert the gene fragment of the recombinant human type III collagen into the expression plasmid of the expression host, transform the expression host, and screen for high-expression recombinant engineered bacteria.

[0009] Specifically, the expression host is Escherichia coli BL21 (DE3).

[0010] Specifically, the expression plasmid is N3A-TEV-8HIS-pET-28A(+), and its nucleotide sequence is shown in SEQ ID No. 4. The coding gene N3Aaa155-377 of the expression plasmid N3A-TEV-8HIS-pET-28A(+) is double-digested with 5'NcoI and 3'EcoRI and connected to pET-28A(+).

[0011] Furthermore, the present invention provides a recombinant human type III collagen expressed by the above expression system.

[0012] Furthermore, the present invention also claims protection for biomedical materials, cosmetics or skin care products comprising the recombinant human type III collagen.

[0013] The present invention selects the fragment sequence of the N-terminal domain and the collagen triple repeat region connected thereto (amino acids 155-377) from the mature peptide chain of the human type III collagen subunit α1-III of known sequence, with a total of 223 amino acids, of which the first 13 amino acids belong to the N-terminal domain of the non-triple helical functional region, which can play a head traction role, making the 14-223 amino acids less likely to mismatch or entangle, helping the collagen peptide subunit monomer to form a helical structure, and enhancing the hydrophilic properties and expression efficiency of the collagen peptide as a whole. An 8-His histidine tag is added to the rear end, and a TEV enzyme cleavage site is inserted between the collagen sequence and the histidine tag to facilitate the later purification to obtain a protein sample with high purity requirements. The gene fragment is inserted into an Escherichia coli expression plasmid, and the Escherichia coli is transformed and screened to obtain a highly expressed recombinant Escherichia coli engineered bacterium. After preliminary fermentation and purification steps, a high-purity recombinant human collagen is obtained.

[0014] Compared with the prior art, the present invention has the following beneficial effects or advantages:

[0015] The recombinant human type III collagen provided by the present invention has good hydrophilicity and stability, and has high sequence homology. Its gene sequence is 100% identical to the corresponding part of the human type III collagen gene sequence, and has application potential in the fields of biomedical materials, beauty cosmetics and skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1This is an amino acid hydrophobicity analysis diagram of human type III collagen. The left picture is the original fragment of human type III collagen subunit α1-III collagen, and the right picture is recombinant human type III collagen.

[0017] Figure 2 Schematic diagram of the construction of recombinant human type III collagen expression plasmid.

[0018] Figure 3 This is a plate image of E. coli DE3 after transformation.

[0019] Figure 4 These are the immunoblotting results of a small-scale expression assay of recombinant human type III collagen. The upper figure shows the protein expression results at different inducer concentrations, and the lower figure shows the protein expression results at different temperatures.

[0020] Figure 5 The figure shows the results of purification and identification of recombinant human type III collagen. The upper figure is the protein gel electrophoresis figure, and the lower figure is the immunoblotting result figure; among them, M: protein standard; 1: whole bacteria before induction; 2: whole bacteria after induction; 3: whole bacteria after disruption; 4: supernatant 1 of centrifugation after disruption of bacteria; 5: centrifugal precipitate 1 of centrifugation after disruption of bacteria; 6: supernatant 2 of centrifugation after disruption of bacteria; 7: centrifugal precipitate 2 of centrifugation after disruption of bacteria; 8: supernatant before salting out; 9: supernatant after salting out; 10: supernatant after acid precipitation and dialysis; B: column material after affinity chromatography; E: affinity chromatography eluate.

[0021] Figure 6 This is the result of mass spectrometry identification of recombinant human type III collagen purification.

[0022] Figure 7 This is the Fourier transform infrared spectroscopy absorption result of recombinant human type III collagen.

[0023] Figure 8 This is the UV absorption spectrum of recombinant human type III collagen.

[0024] Figure 9 This is the circular dichroism spectrum result of recombinant human type III collagen.

[0025] Figure 10 This is the scanning electron microscopy result of recombinant human type III collagen. Specific embodiments

[0026] The technical solutions of the present invention are described below with reference to the following embodiments; however, the present invention is not limited to the following embodiments.

[0027] The experimental methods and detection methods in the following embodiments are all conventional methods unless otherwise specified; the agents and materials are all commercially available unless otherwise specified; the indicator data are all based on conventional measurement methods unless otherwise specified.

[0028] Example 1

[0029] 1. Protein sequence selection

[0030] Based on the analysis of the amino acid sequence and structural region of the human type III collagen subunit α1-III chain collagen, the amino acid sequence of the present invention (SEQ ID No. 2) selected the N-terminal domain of the human type III collagen subunit α1-III and the fragment sequence of the collagen triple repeat region connected thereto (amino acids 155-377), a total of 223 amino acids. Among them, the first 13 amino acids belong to the N-terminal domain of the non-triple helical functional region, which can play a head traction role, making the 14th to 223 amino acids less likely to mismatch or entangle, helping the collagen peptide subunit monomer to form a helical structure, and enhancing the overall hydrophilicity and expression efficiency of the collagen peptide. An 8-His histidine tag is added to the back end, and a TEV enzyme cleavage site is inserted between the collagen sequence and the histidine tag to facilitate the acquisition of recombinant collagen samples with high purity requirements and no affinity tags by affinity purification and enzyme cleavage. The amino acid sequence of the selected human type III collagen subunit α1-III chain collagen and the recombinant human type III collagen sequence with a histidine tag were subjected to hydrophobicity analysis, and the evaluation results were as follows: Figure 1 As shown, the hydrophobicity of most amino acids in the recombinant collagen sequence is lower than zero, indicating good hydrophilicity.

[0031] 2. Plasmid Construction

[0032] The plasmid N3A-TEV-8HIS-pET-28A(+) of the present invention is designed by ourselves. The schematic diagram of plasmid construction is shown in FIG. Figure 2 As shown, the plasmid encoding gene N3Aaa155-377 was obtained by reverse translation of the protein sequence and synthesized after codon optimization in E. coli. Its nucleotide sequence is shown in SEQ ID No. 4. It was double-digested with 5' NcoI and 3' EcoRI and then ligated into pET-28A(+). Gene synthesis was performed by Jiangsu GenScript Biotech.

[0033] 3. Transformation of Escherichia coli BL21(DE3)

[0034] The lyophilized plasmid powder was dissolved in sterile water to 100 ng / μL, 10 μL of the dissolved plasmid was added to a 1.5 mL EP tube containing 100 μL of E. coli DE3 competent cells, placed on ice for 30 min, then heat-shocked in a 42°C water bath for 90 s, and then placed on ice for 2-3 minutes. Then, 800 μL of LB liquid culture medium was added and shake-cultured at 37°C for 1 h. Finally, 100 μL of the liquid was spread on a solid culture medium containing kanamycin resistance and cultured at 37°C overnight. Figure 3This is a plate image of E. coli DE3 after transformation.

[0035] 4. Small-scale expression identification

[0036] Two groups of experiments were conducted to screen the inducer concentration and induction expression temperature conditions and to identify the expression. 20 μL of the transformed E. coli strain was inoculated into 50 mL of LB conical flask (a total of 19 copies) and cultured at 220 rpm and 37 ° C until the OD 600 =0.6, group 1 was induced with 0.1-1mM IPTG and cultured at 37°C and 160rpm for 24h; group 2 was induced with 1mM IPTG+30°C, 0.5mM IPTG+30°C, and 1mM IPTG+16°C, and samples were taken at 2, 4, 6 hours, 19, 23, and 25 hours of induction. After the culture was completed, cells were collected at 6000rpm, 10min, and 4°C, and the histidine tag was detected by protein immunoblotting after whole-cell protein gel electrophoresis to determine whether the protein was expressed. The immunoblotting results of the small-scale expression identification are as follows: Figure 4 , where the upper figure shows the protein expression results at different inducer concentrations, and the lower figure shows the protein expression results at different temperatures. Figure 4 It can be seen that the recombinant protein was well expressed under 0.1-1 mM IPTG conditions, and the expression yield was significantly improved at 16°C.

[0037] 5. A method for purifying and preparing recombinant human type III collagen, comprising the following steps:

[0038] (1) Bacterial disruption and centrifugation

[0039] Recombinant Escherichia coli capable of expressing recombinant human type III collagen was dissolved in a buffer solution (Tris-HCl, EDTA), and the bacteria were broken using a homogenizer. The supernatant was then collected by centrifugation.

[0040] (2) Salting out

[0041] Add solid NaCl at 10 g / L to the supernatant obtained by centrifugation, gradually adding while stirring, so that some impurity proteins can precipitate, and collagen is still preserved in the supernatant at this NaCl concentration), centrifuge, and then take the supernatant.

[0042] (3) Acid precipitation

[0043] The pH value of the centrifugal supernatant containing NaCl is adjusted to between 2.0 and 4.5 (when the pH value is between 2.0 and 4.5, impurity proteins can be further precipitated, and the target collagen is preserved in the supernatant without precipitation). Centrifuge and retain the supernatant.

[0044] (4) Dialysis

[0045] The supernatant obtained after acid precipitation and centrifugation is dialyzed (using the same concentration of dialysis buffer as for lysis). This serves two purposes: first, to remove any added NaCl from the supernatant; second, to adjust the pH of the supernatant to that of the buffer. The dialysis buffer is replaced several times until the internal and external conductivity and pH are consistent.

[0046] (5) Affinity chromatography

[0047] After adding 20 mL of ddH2O to the Ni affinity chromatography column for cleaning, slowly add the supernatant containing the affinity protein along the column wall. After the sample is loaded, rinse with lysis buffer salt solution to remove impurities, and use imidazole eluent to competitively elute the protein bound to the Ni affinity column material.

[0048] (6) Ion exchange and reverse ion pair chromatography

[0049] If further purity improvement is required, the affinity chromatography eluate can be eluted through a Sp-Sepharose.FF (GE) chromatography column. After the collagen target peak is collected, the endotoxin in the collected liquid can be completely removed by passing it through a reverse C18 column.

[0050] 6. Identification and Analysis

[0051] (1) SDS-PAGE electrophoresis detection:

[0052] Collect the sample and add 5× loading buffer, heat in a 100℃ metal bath for 10 minutes; take 10μL of the sample and load it on a 15% polyacrylamide gel electrophoresis; remove the gel and place it in a plastic container, cut off the concentrated gel part, add an appropriate amount of staining solution to cover the gel; place it on a shaker and shake it, and obvious bands will appear in about 5-10 minutes.

[0053] The SDS-PAGE results of the purified recombinant human type III collagen expressed by the engineered bacteria are as follows Figure 5 (Top) After the purification process, the recombinant protein was significantly enriched and its purity gradually improved. The molecular weight of the band on the gel was slightly higher than the predicted molecular weight of the recombinant protein. M: Protein standard; 1: Whole bacteria before induction; 2: Whole bacteria after induction; 3: Whole bacteria after disruption; 4: Supernatant 1 after disruption (3000 rpm, 15 min); 5: Centrifugation 1 after disruption (3000 rpm, 15 min); 6: Supernatant 2 after disruption (13000 rpm, 60 min); 7: Centrifugation 2 after disruption (13000 rpm, 60 min); 8: Supernatant before salting out; 9: Supernatant after salting out; 10: Supernatant after acid precipitation and dialysis; B: Column material after affinity chromatography; E: Eluate from affinity chromatography.

[0054] (2) Western Blotting

[0055] The samples were subjected to polyacrylamide gel electrophoresis. After the electrophoresis, the gel was removed and transferred to a membrane at a constant voltage of 90 V for 90 minutes. The membrane was blocked in 5% skim milk powder at room temperature for 60 minutes. The primary antibody was diluted with antibody diluent, and the membrane was incubated in the primary antibody diluent (dilution ratio 1:1000) at 4°C overnight. The next day, the membrane was removed and washed three times with PBST for 10 minutes each time. The secondary antibody was diluted with antibody diluent, and the membrane was reacted in the secondary antibody diluent (dilution ratio 1:1000) at room temperature for 1 hour. After the reaction was completed, the membrane was removed and placed in a clean box and washed three times for 10 minutes each time. ECL development and exposure were performed.

[0056] The results of protein immunoblotting of purified recombinant human type III collagen expressed by engineering bacteria are as follows Figure 5 (Bottom) Compared with the SDS-PAGE results, the protein enriched in the affinity chromatography eluate is the target recombinant human type III collagen.

[0057] (3) Protein gel mass spectrometry analysis:

[0058] ① The protein gel was stained with Coomassie Brilliant Blue R-250 solution;

[0059] ②According to the collagen composition unit, the subunit band corresponding to its molecular weight is removed and transferred to the EP tube;

[0060] ③Protease digestion: cut the gel band into pieces of about 1mm 3 The sections were mixed with 300 μL of 100 mmol / mL NH₄CO₃ in 30% acetonitrile. After removing the supernatant, 50 μL of acetonitrile and 5 μL of 5 ng / μL trypsin were added, and the mixture was incubated at 4°C for 30 minutes. After incubation, the remaining liquid was removed, and 20 μL of 25 mmol / L NH₄CO₃ was added. The mixture was incubated at 37°C overnight, after which the solution was transferred to a fresh tube and centrifuged.

[0061] ④ Protein primary mass spectrometry identification: MALDI-TOF / TOF is used for detection to obtain PMF peptide mass fingerprints, which are matched with the theoretical peptide masses in the database for protein identification;

[0062] ⑤ Protein identification by secondary mass spectrometry (tandem mass spectrometry): Identification was performed using a super-transmitter MALDI-TOF / TOF mass spectrometer equipped with Mascot sequence software containing the NCBI database.

[0063] Protein gel mass spectrometry results of affinity chromatography eluate are as follows Figure 6Through the confidence, relative content search ranking and mass spectrometry identification of the comparison results of the polypeptide sequence and the target sequence, it can be determined that the final purified protein is the target recombinant human type III collagen.

[0064] 7. Advanced structural characterization methods for recombinant human type III collagen:

[0065] (1) Fourier transform infrared spectroscopy (FT-IR)

[0066] Take the freeze-dried protein sample and press it into a uniform film, then put it into a desiccator filled with P2O5 for later use. Scan it with a Fourier transform infrared spectrometer at room temperature and record the wavelength of 4000-5000 cm -1 Spectrum of the interval.

[0067] The FT-IR results of recombinant human type III collagen lyophilized powder are as follows Figure 7 As shown, it contains all the characteristic peaks of collagen. The amide I band is related to the C=O stretching vibration of the peptide chain. This peak has the strongest absorption, indicating that the protein is in a cross-linked state. The amide II band is caused by the coupling of NH bending vibration and CN stretching vibration. The amide A band is related to the stretching vibration of NH in collagen and the hydrogen bond formed by the carbonyl group. Both of them have a slight blue shift, indicating the presence of hydrogen bonds in the protein. The peak of the amide III band is at 1400-1454 cm -1 The peak ratio within the range is very close to 1, indicating that the protein has a triple helical structure.

[0068] (2) Ultraviolet absorption spectrum

[0069] The lyophilized protein sample was prepared into a 0.9 mg / mL protein solution and placed in a sample cell with an optical path of 1 cm for scanning analysis at a scanning wave number of 200-400 nm and room temperature.

[0070] The UV absorption spectrum of recombinant human type III collagen is as follows Figure 8 As shown, the maximum absorption peak is located at 223 nm, which is mainly related to the distinct absorption peaks of chromophores such as -C=O, -COOH, and CO-NH2 contained in the peptide chain. A weaker absorption peak is also observed at 275 nm. These results are consistent with the UV absorption characteristics of type III collagen.

[0071] (3) Circular dichroism

[0072] Take an appropriate amount of collagen sample and dissolve it in a buffered saline solution to prepare a solution with a concentration of 0.1 mg / mL. Take a small amount of the above solution and place it in a sample cell with an optical path of 1 mm for scanning analysis. The scanning wave number is 190-250 nm and the temperature is room temperature.

[0073] The circular dichroism results of recombinant human type III collagen are as follows Figure 9 As shown, the protein has an obvious negative absorption peak near 198nm, a weaker positive absorption peak at 223nm, and a cross point at 217nm, showing the typical characteristics of the collagen triple helix structure.

[0074] (4) Scanning electron microscopy

[0075] An appropriate amount of collagen sample was placed on the stage, and after ion sputtering gold treatment, the collagen microstructure was observed using a scanning electron microscope at an accelerating voltage of 10.0 kV and a magnification of 100-1000 times.

[0076] Scanning electron microscope image of recombinant human type III collagen Figure 10 As shown, the outer layer of collagen can form a dense reticular sheet structure ( Figure 10 , above), the reticular structure is made of interwoven protein fibers ( Figure 10 , middle), individual collagen fibers can be seen ( Figure 10 ,Down).

[0077] In summary, the purification method of recombinant human type III collagen provided by the present invention uses a homogenizer to break the bacteria so that the lysis rate of Escherichia coli reaches more than 98%, thereby minimizing the loss of bacteria and protein in the lysis step of recombinant human type III collagen. Salting out and acid precipitation respectively use the principle of different solubility of proteins in different salt concentrations and different isoelectric points of proteins to remove foreign proteins, thereby not affecting the activity of recombinant human type III collagen. Combined with affinity chromatography and ion exchange chromatography to effectively prevent collagen molecules from decomposing into small molecular peptides, and reverse chromatography purification technology to remove heat sources, the purified recombinant human type III collagen is a high-purity collagen that meets the requirements of cosmetic raw materials, food grade and medical device grade.

[0078] The recombinant human type III collagen expressed by the engineered bacteria constructed using this method was tested by protein gel electrophoresis and immunoblotting to determine the protein's molecular weight and purity; circular dichroism, Fourier transform infrared spectroscopy, and ultraviolet spectroscopy confirmed that the protein had the characteristic structure of collagen; and scanning electron microscopy confirmed that the protein had the advanced three-dimensional structure and potential support and elastic properties of collagen, indicating its application value.

[0079] As described above, the present invention can be better implemented. The above embodiments only describe the preferred implementation methods of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the present invention.

Claims

1. A recombinant human type III collagen, characterized in that: The amino acid sequence of the recombinant human type III collagen is shown in SEQ ID No.

2.

2. The recombinant human type III collagen according to claim 1, characterized in that The recombinant human type III collagen encoding gene N3Aaa155-377, the nucleotide sequence of the gene N3Aaa155-377 is shown in SEQ ID No.

3.

3. A method for expressing recombinant human type III collagen according to any one of claims 1 to 2, characterized in that: The construction method of the expression method is to insert the gene fragment of the recombinant human type III collagen into the expression plasmid of the expression host, transform the expression host, and screen to obtain high-expression recombinant engineering bacteria.

4. The expression method according to claim 3, characterized in that The expression host is Escherichia coli BL21 (DE3).

5. The expression method according to claim 3, characterized in that The expression plasmid is N3A-TEV-8HIS-pET-28A(+).

6. The expression method according to claim 5, characterized in that The nucleotide sequence of the expression plasmid N3A-TEV-8HIS-pET-28A(+) is shown in SEQ ID No.

4.

7. The expression method according to claim 5, characterized in that The coding gene N3Aaa155-377 of the expression plasmid N3A-TEV-8HIS-pET-28A(+) is double-digested with 5'NcoI and 3'EcoRI and then connected to pET-28A(+).

8. A recombinant human type III collagen, characterized in that: The method is expressed according to any one of claims 3 to 7.

9. A biomedical material, cosmetic or skin care product, characterized in that: Contains the recombinant human type III collagen according to claim 8.

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