Recombinant human collagen and preparation method thereof

Abstract

The present invention provides a recombinant human type III collagen and a method for its construction. The recombinant human type III collagen comprises 468 amino acids, with a basic repeating unit of GERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGER. The recombinant human collagen of the present invention is 100% identical to the corresponding portion of the natural collagen gene sequence, has a triple-helical structure, and exhibits excellent hydrophilicity and stability. The production steps include: construction of genetically engineered Escherichia coli bacteria, fermentation and cultivation of the genetically engineered bacteria, and purification of the recombinant human collagen. The present invention does not cause immune rejection when used in the human body and can be widely used in biomedical materials, cosmetics, and other fields.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and relates to human type III recombinant human collagen and a production method. Background Art

[0002] Collagen is a biopolymer widely found in animal connective tissue, accounting for approximately 25%-30% of the total protein in the body. It is the most widely distributed and most abundant functional protein in mammals. Due to its excellent bioactivity, biocompatibility, and biodegradability, it has a wide range of applications in biomedicine, food, and cosmetics.

[0003] However, natural collagen is insoluble in water and difficult to be used by the human body. It requires special chemical treatment before use. Currently, most collagen on the market is extracted from terrestrial animal connective tissue and aquatic product processing by-products through hot water extraction, acid-base hydrolysis, and enzymatic hydrolysis. However, the separation and purification process of animal-derived collagen is complex and monomer separation is difficult. It may also carry viruses, posing a safety hazard. At the same time, it is incompatible with the human body and can cause severe immune rejection reactions. Therefore, animal-derived collagen can only be used in cosmetics and health products, and its original biological functions cannot be realized in the human body.

[0004] In recent years, the use of genetic engineering to produce collagen has rapidly developed, with research on this topic becoming increasingly in-depth. The challenges of producing collagen using genetic engineering techniques lie in achieving a correct triple helical structure, improving water solubility, and adapting it for large-scale production. Natural collagen molecules are left-handed helices based on a repeating structure of three amino acid residues (Gly-XY), forming a unique superhelical structure. Proline (Pro) and hydroxyproline (Hyp) occupy a high proportion of the X and Y positions, accounting for approximately 25%, the highest concentration among all proteins. In organisms, collagen synthesis and modification begins with tropocollagen. Through the complex regulation of multiple enzymes, it undergoes numerous chemical changes, including hydroxylation, glycosylation, and cross-linking, to form biologically active collagen. In terms of protein structure, 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. Therefore, collagen produced according to the original genetic sequence struggles to spontaneously organize into the correct spatial structure in vitro. Such difficulties severely hampered the development and production of human collagen. Subsequently, researchers attempted to increase proline hydroxylation levels by introducing proline hydroxylase (P4H) in order to achieve a correct triple-helical structure. Studies have shown that even when P4H activity in transgenic silk glands was 130 times higher than that of wild-type, the resulting human type I collagen still failed to form a correct triple-helical structure. Co-expression of type III collagen and P4H using an insect cell expression system resulted in a stable triple-helical structure, but the expression level was only 50 mg / L. Therefore, expressing collagen in animal and plant cells is difficult and costly, making it unsuitable for large-scale production. The study found that the collagen obtained by co-expressing P4H in Pichia pastoris can fold into the correct spatial structure and can be fully hydroxylated, and the expression level can reach 0.2~0.6g / L, but it cannot be secreted outside the cell and can only accumulate in the endoplasmic reticulum lumen of the cell; Gao Lihu et al. designed and synthesized a gene monomer encoding a human collagen protein with a highly hydrophilic Gly-XY tripeptide repeat sequence based on the Gly-XY tripeptide repeat sequence characteristics of the collagen domain of the α1 chain of human type III collagen, with the purpose of improving the water solubility of collagen and increasing the expression level, and then constructed a high-repeat sequence human collagen protein expression vector through tandem in the same direction, and finally transformed it into Pichia pastoris, realizing the secretory expression of recombinant human collagen protein, but it did not generate a collagen trimer structure. Previous studies have suggested that the inability to form collagen trimers is due to the lack of head and tail structures. Therefore, some researchers have attempted to enhance the gel-forming properties of truncated Gly-XY tripeptide repeats by adding the hinge region amino acids GPPGPCCGGG to the C-terminus of the peptide (Journal of Biochemistry, 2004; 136: 643-649).However, in actual implementation, the addition of a GPPGPCCGGG linker to the C-terminus of the peptide results in the formation of a colloidal precipitate in most target peptides during shake flask or fermentation culture, making it difficult to dissolve and purify. This results in a very low yield and prohibits large-scale production. Jinbo Bio has disclosed a collagen peptide sequence that can form a stable trimer structure even without the GPPGPCCGGG linker (reference patent CN109593126A), demonstrating that the appropriate Gly-XY tripeptide repeat sequence can achieve complete collagen structure and function when recombinantly expressed in vitro. However, the collagen expression level is low, and the peptide contains only the GER tripeptide at the C-terminus, which may result in poor hydrophilicity and requires further improvement in achieving complete collagen function. Summary of the Invention

[0005] The present invention provides the following:

[0006] The purpose of the present invention is to provide a stable recombinant human collagen and a production method.

[0007] The technical solution adopted in the present invention is:

[0008] 1. Recombinant human collagen, wherein the basic repeating unit is the polypeptide sequence of GERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGER shown in SEQ ID No.1.

[0009] 2. The polypeptide according to item 1, comprising n repeats of the sequence shown in SEQ ID No. 1, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24, or 32, wherein when n is an integer greater than or equal to 2, the repeated sequences are directly linked.

[0010] 3. The polypeptide according to item 2, wherein the polypeptide comprises:

[0011] 1) the amino acid sequence of SEQ ID No. 2;

[0012] 2) an amino acid sequence that is 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID No. 2, which retains the triple helical structure of the amino acid sequence of SEQ ID No. 2;

[0013] 3) An amino acid sequence in which one or more amino acids are added, substituted, or deleted from the amino acid sequence of SEQ ID No. 2 and the protein has the same function, and which retains the triple helical structure of the amino acid sequence of SEQ ID No. 2; or

[0014] 4) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence of SEQ ID No. 2 under stringent conditions, wherein the amino acid sequence retains the triple helical structure of the amino acid sequence of SEQ ID No. 2, wherein the stringent conditions are moderately stringent conditions, moderate-high stringency conditions, high stringency conditions, or very high stringency conditions.

[0015] 4. The DNA sequence encoding the recombinant human collagen C3-15 shown in SEQ ID NO. 2 of the present invention is shown in SEQ ID NO. 3.

[0016] When the recombinant human collagen of the present invention is expressed, a six-histidine tag is added to its N-terminus and connected via an ENLYFQ (SEQ ID No. 4) sequence, which can be directly removed by TEV protease to obtain the sequence of SEQ ID No. 2.

[0017] 5. The present invention provides a recombinant vector comprising the nucleic acid of the present invention.

[0018] In the present invention, the recombinant vector further comprises a backbone vector; wherein the backbone vector is preferably pET32a.

[0019] 6. The present invention also provides a host transformed with the recombinant vector.

[0020] In the present invention, the host is Escherichia coli, specifically Escherichia coli BL21.

[0021] 7. The present invention provides a method for producing recombinant human collagen.

[0022] The method for producing recombinant human collagen of the present invention comprises the following steps:

[0023] 1) Construction of genetically engineered Escherichia coli;

[0024] 2) Fermentation culture of genetically engineered Escherichia coli;

[0025] 3) Inducible expression of recombinant human collagen;

[0026] 4) Purification of recombinant human collagen.

[0027] In the present invention, the purification method is Ni ion (nickel ion) affinity chromatography.

[0028] The steps for constructing the genetically engineered Escherichia coli described in step 1) are as follows: (1) selecting a DNA fragment of the helical region of the human type III collagen gene, codon-optimizing the fragment, and obtaining a complete recombinant gene by gene synthesis; (2) cloning the DNA fragment using the expression vector pET-32a, and transforming it into BL21 (DE3) Escherichia coli to obtain the genetically engineered Escherichia coli by screening.

[0029] The fermentation culture of the genetically engineered E. coli in step 2) is as follows: a single colony of the genetically engineered E. coli obtained by screening is inoculated into 5 ml of LB culture medium and cultured at 37° C. overnight.

[0030] The fermentation culture of the genetically engineered Escherichia coli described in step 3) is as follows: the bacterial solution is inoculated into 2YT medium at a 1% inoculum, cultured at 37°C until the OD600 is between 0.4 and 0.6, 0.5 mM IPTG is added to induce expression, the temperature is lowered to 16°C, and the culture is continued at 120 rpm for 15 hours, and the bacteria are collected by centrifugation.

[0031] The purification of the recombinant human collagen described in step 4) is as follows: (1) resuspending the bacteria with nickel column equilibrium solution, ultrasonically disrupting them, and collecting the supernatant by centrifugation; (2) purifying the recombinant human collagen from the supernatant using nickel ion affinity chromatography; and (3) cleaving the recombinant human collagen using TEV protease.

[0032] Advantages of the recombinant collagen of the present invention: (1) The recombinant human collagen produced by the collagen sequence provided by the present invention is 100% identical to the corresponding portion of the natural collagen gene sequence, has a triple helical structure, good hydrophilicity and stability, strong cell adhesion activity, and high protein expression. (2) The use of an E. coli expression system shortens the culture time and is low-cost, making it suitable for large-scale industrial production. (3) Six histidine-specific affinity purification tags are attached to the N-terminus, resulting in a high-purity product. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Hydrophilicity prediction map of C3-15 recombinant human type III collagen;

[0034] Figure 2 :Purification and stability of C3-15 recombinant human type III collagen;

[0035] Figure 3 : C3-15 recombinant human type III collagen triple helix structure;

[0036] Figure 4 : Thermal stability of C3-15 recombinant human type III collagen;

[0037] Figure 5 :Cell adhesion activity of C3-15 recombinant human type III collagen. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the present invention more clear, the invention is clearly and completely described below in conjunction with specific embodiments.

[0039] Example 1 Gene design and expression

[0040] 1. Human collagen C3-15 gene design and synthesis

[0041] (1) Gene design: A polypeptide fragment containing 30-40 amino acids starting or ending with GER in the helical region of the human type III collagen gene was selected as the repeating unit, and the sequence was shown in SEQ ID No. 1. The hydrophilicity of the above polypeptide sequence was analyzed using the online tool ProtScale for protein hydrophilicity mapping, and sequences with a high hydrophilicity index were selected for gene synthesis.

[0042] (2) Gene synthesis: The collagen repeat sequence SEQ ID No. 1 was repeated 12 times to obtain the amino acid sequence SEQ ID No. 2 of recombinant human collagen C3-15. The DNA sequence SEQ ID No. 3 of recombinant human collagen C3-15 was submitted to GenScript Biotech Co., Ltd. for full gene synthesis and cloned into the pET32a vector.

[0043] 2. Construction of Engineered Bacteria

[0044] The synthesized C3-15-pET32a plasmid powder was centrifuged and dissolved in 40 μL sterile water. 3 μL of the solution was added to the BL21 (DE3) competent cells and gently mixed. The cells were placed on ice for 30 minutes. Heat-shocked in a 42°C water bath for 90 seconds, the cells were immediately placed on ice for cooling. 500 μL of liquid LB culture medium was added and mixed. The cells were placed on a 37°C shaker for recovery for 1 hour. The transformed cells were spread on an LB plate (containing 50 μg / ml kanamycin), and the cells were inverted and cultured overnight in a 37°C incubator. The grown colonies were genetically engineered Escherichia coli.

[0045] 3. Induced Expression of Engineered Bacteria

[0046] Single clones were picked from the transformed plates and cultured overnight in 5 ml of LB medium (containing 50 μg / ml kanamycin). The bacterial suspension was inoculated into 2YT medium (16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride) at a 1% inoculum size for expansion. The culture was cultured at 37°C until the OD600 was between 0.4 and 0.6. IPTG was added to a final concentration of 0.5 mM to induce expression. The temperature was lowered to 16°C and the culture was continued at 120 rpm for 15 h. The bacteria were then collected by centrifugation.

[0047] 4. Purification of recombinant human collagen.

[0048] (1) Bacterial disruption: Resuspend 100 ml of bacterial pellet in 5 ml of equilibrium solution (20 mM phosphate, 200 mM sodium chloride, 10 mM imidazole, pH 8.0), disrupt the cells by ultrasonication, and centrifuge at 12,000 rpm for 10 min. Keep the supernatant.

[0049] (2) Protein purification: Use 5 column volumes of equilibration solution to equilibrate a HisTrap HP affinity column (Cytiva). The sample supernatant is filtered through a 0.22 μm filter membrane and then loaded. After loading, rinse with equilibration solution and then wash with washing solution (20 mM phosphate, 200 mM sodium chloride, 40 mM imidazole, pH 8.0) to remove impurities. Add TEV protease and digest on the column at 4°C for 16 h to obtain the target collagen peptide. The collected target protein is exchanged into Tris-HCl buffer and stored at 4°C.

[0050] 5. Protein Concentration Detection

[0051] Protein sample concentration was determined using the UV absorption method: UV absorbance was measured at 215 nm and 225 nm, and the protein concentration was calculated using the empirical formula C (μg / ml) = 144 × (A215 - A225). Note that A215 should be < 1.5. This method, which measures the characteristic absorption of peptide bonds under far-UV light, is unaffected by chromophore content, exhibits minimal interfering substances, and is simple to use. It is suitable for detecting human collagen and its analogs, which are not colorimetrically sensitive to Coomassie Brilliant Blue. For recombinant human collagen C3-15, 250 ml of bacterial culture yields 750 mg of purified protein.

[0052] Example 2 Detection of activity of recombinant human collagen

[0053] 1. Prediction of hydrophilicity of C3-15

[0054] The hydrophilicity prediction of the amino acid sequence of SEQ ID No. 2 was performed using the online tool ProtScale (https: / / web.expasy.org / protscale / ) for protein hydrophilicity analysis. The results are as follows: Figure 1 As shown, there is basically no hydrophobic peak, indicating that the predicted results of the C3-15 amino acid sequence are highly hydrophilic and have no hydrophobic region.

[0055] 2. Purity of C3-15 recombinant human collagen was tested using SDS-PAGE

[0056] Sample treatment: 45 μl of sample was added to 15 μl of 4× loading buffer, mixed evenly, and incubated in a boiling water bath for 10 min. The mixture was then cooled naturally for later use.

[0057] Protein electrophoresis: Samples were loaded onto GenScript SurePAGE™ precast gels (4-12%) and run at 140V for approximately 90 minutes until the bromophenol blue band reached the bottom of the gel. Protein staining was then performed using a GenScript eStain® L1. Due to the unique amino acid sequence and structural characteristics of collagen, its migration speed is relatively slow. See the results for details. Figure 2 In the second lane, the purified C3-15 recombinant humanized collagen was a single band, indicating that the recombinant humanized collagen was successfully prepared.

[0058] 3. Stability test of C3-15 recombinant human collagen

[0059] The C3-15 protein was stored in Tris-HCl buffer at 4°C and its purity was detected by SDS-PAGE after 1 week, 2 weeks, 3 weeks, and 6 weeks. Figure 2 As shown in the third, fourth, fifth and sixth lanes, the C3-15 protein did not undergo obvious degradation after 1 week, 2 weeks, 3 weeks and 6 weeks, indicating that the C3-15 recombinant human collagen has good stability.

[0060] 4. Circular Dichroism Characterization of C3-15 Recombinant Human Collagen

[0061] Circular dichroism is a common method for characterizing protein structure. The positive peak near 225nm is the characteristic peak of the collagen triple helix structure. Circular dichroism was used to identify C3-15 recombinant humanized collagen. The results are as follows: Figure 3 As shown in . The protein has a positive peak at 225 nm, indicating that C3-15 recombinant humanized collagen has a triple helical structure.

[0062] 5. Differential Scanning Calorimetry Analysis of C3-15 Recombinant Humanized Collagen

[0063] Differential scanning calorimetry was used to analyze C3-15 recombinant humanized collagen. Figure 4 As shown in , there is an obvious characteristic peak at 33.5°C, that is, the protein transforms from an ordered triple helix structure to a disordered structure, and the Tm value of C3-15 recombinant humanized collagen is 33.5°C.

[0064] VI. Adhesion testing of C3-15 recombinant humanized collagen

[0065] Use PBS to adjust the protein concentration of C3-15 recombinant humanized collagen and Sigma human collagen type III protein (control group) to 0.5 mg / ml, add 100ul of the test protein solution and blank PBS solution control to the 96-well plate, and let it stand at room temperature for 60 minutes; add 105 3T3 cells to each well and incubate at 37°C for 60 minutes; wash each well 4 times with PBS; use the LDH detection kit to detect the absorbance at OD492nm. Based on the value of the blank control, the cell adhesion rate can be calculated. The calculation formula is as follows: Cell adhesion rate = (test well - blank well) × 100% / (positive well - blank well). Figure 5 As shown in the figure, the cell adhesion rate of C3-15 is better than that of the control group, indicating that C3-15 has high activity and can provide cells with a high-quality external environment in a short time to help cells adhere to the wall.

Claims

1. A recombinant human collagen, characterized in that: The structure of the recombinant human collagen is triple helical, and its basic repeating unit is shown in SEQ ID No. 1: GERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGER, which is a human collagen type III peptide segment; The recombinant human collagen is C3-15, and the amino acid sequence is shown in SEQ ID No.

2.

2. The nucleic acid encoding the recombinant human collagen C3-15 according to claim 1, the DNA sequence of which is shown in SEQ ID No.

3.

3. A recombinant vector comprising the nucleic acid according to claim 2. A host cell comprising the recombinant vector according to claim 3.

5. The host cell according to claim 4, characterized in that The host cell is Escherichia coli.

6. The method for producing recombinant human collagen according to claim 1, characterized in that: (1) Transforming Escherichia coli with the recombinant vector according to claim 3 to obtain genetically engineered Escherichia coli; (2) Fermentation culture of genetically engineered Escherichia coli; (3) Inducible expression of recombinant human collagen; (4) Purification of recombinant human collagen.

7. The method for producing recombinant human collagen according to claim 6, characterized in that: The steps for constructing the genetically engineered Escherichia coli described in step (1) are as follows: a. Select a DNA fragment of the helical region of the human type III collagen gene, perform codon optimization on the fragment, and obtain a complete recombinant gene by gene synthesis; b. Use the expression vector pET-32a to clone the DNA fragment and transform it into BL21 (DE3) Escherichia coli to screen and obtain genetically engineered Escherichia coli bacteria.

8. The method for producing recombinant human collagen according to claim 6, characterized in that: The induced expression of the recombinant collagen described in step (3) is as follows: the bacterial solution is inoculated into 2YT medium at a 1% inoculum volume, cultured at 37°C until the OD600 is between 0.4 and 0.6, 0.5 mM IPTG is added to induce expression, the temperature is lowered to 16°C, and the culture is continued at 120 rpm for 15 hours, and the bacteria are collected by centrifugation.

9. The method for producing recombinant human collagen according to claim 6, characterized in that: The purification of the recombinant collagen described in step (4) is as follows: a. Resuspend the bacteria in nickel column equilibration solution, disrupt with ultrasound, and collect the supernatant by centrifugation; b. Purifying recombinant human collagen from the supernatant using nickel ion affinity chromatography; c. Use TEV protease to cleave recombinant human collagen.

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