Recombinant human collagen and preparation method thereof

Abstract

The invention provides a recombinant human-derived III-type collagen and a construction method of the recombinant human-derived III-type collagen. The recombinant human source III type collagen comprises 468 amino acids, and a basic repeating unit of the recombinant human source III type collagen is GERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGER. The recombinant human source III type collagen comprises 468 amino acids, the recombinant human collagen provided by the invention is 100% identical to a corresponding part of a natural collagen gene sequence, has a triple helix structure, and has good hydrophilicity and stability. The production method comprises the following steps: construction of escherichia coli genetically engineered bacteria, and fermentation culture of the genetically engineered bacteria; and purifying the recombinant human collagen. The method does not cause immunological rejection when used in a human body, and can be widely applied to the fields of biomedical materials, cosmetics and the like.

Description

Technical Field

[0001] The 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 that is widely present in animal connective tissues, accounting for about 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 good biological activity, biocompatibility and biodegradability, it is widely used in biomedicine, food and cosmetics.

[0003] However, natural collagen is insoluble in water and difficult to be used by the human body. It needs to be treated with special chemical methods before use. At present, most of the collagen on the market is extracted from the connective tissue of terrestrial animals and by-products of aquatic processing through hot water extraction, acid-base hydrolysis and enzymatic hydrolysis. However, the separation and purification process of animal-derived collagen is complicated and the monomer separation is difficult. It may also carry viruses and pose a safety hazard. At the same time, it is incompatible with the human body and will cause serious 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 production of collagen using genetic engineering technology has developed rapidly, and various related studies have become increasingly in-depth. The difficulty of producing collagen using genetic engineering technology lies in forming a correct triple helix structure, improving water solubility and being suitable for large-scale production. Natural collagen molecules are left-handed helices with three amino acid residues (Gly-XY) as the basic repeating body to form a special superhelical structure. The proportion of proline (Pro) and hydroxyproline (Hyp) at the X and Y positions is very high, accounting for about 25%, which is the highest content among all types of proteins. In organisms, the synthesis and modification of collagen starts from procollagen, through the complex regulation of multiple enzymes, through hydroxylation, glycosylation, cross-linking and many other chemical changes, in order to form biologically active collagen. In terms of protein structure, procollagen contains not only collagen chains, but also spherical heads and tails. Without such heads and tails, the collagen chains will not fold to form the correct triple helix, thus lacking the biological activity of collagen. Therefore, it is difficult for collagen prepared according to the original gene sequence to spontaneously organize and form the correct spatial structure in vitro. Such difficulties have seriously hindered the research and development and production of human collagen. Afterwards, researchers tried to increase the level of proline hydroxylation by introducing proline hydroxylase (P4H) in order to form a correct triple helix structure. Studies have shown that when the activity of P4H in transgenic silk glands is 130 times that of the wild type, the expressed human type I collagen still cannot form a correct triple helix structure; using an insect cell expression system to co-express type III collagen and P4H, although it is a stable triple helix structure, the expression level is only 50 mg / L. Therefore, it is difficult and costly to express collagen through animal and plant cells, which is not conducive to 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 of the cell; Gao Lihu et al. designed and synthesized a gene monomer encoding a human collagen with a highly hydrophilic Gly-XY tripeptide repeat sequence based on the characteristics of the Gly-XY tripeptide repeat sequence in 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 expression vector through tandem connection, and finally transformed it into Pichia pastoris, realizing the secretory expression of recombinant human collagen, but it did not generate a collagen trimer structure. Previous studies have suggested that the inability to generate collagen trimers is due to the lack of head and tail structures, so some researchers have attempted to add the hinge region amino acids GPPGPCCGGG to the C-terminus of the polypeptide to improve the gelation of the truncated Gly-XY tripeptide repeat sequence (reference: Journal Of Biochemistry. 2004; 136: 643-649).However, in the actual implementation process, after adding the GPPGPCCGGG linker to the C-terminus of the polypeptide, most of the target polypeptides form a colloidal precipitate during shake flask or fermentation culture, which cannot be dissolved and purified, and the yield is very low, and it cannot be produced on a large scale. Jinbo Bio has disclosed a collagen polypeptide sequence that can still form a stable trimer structure in the absence of the GPPGPCCGGG linker (reference patent CN109593126A), which shows that the appropriate Gly-XY tripeptide repeat sequence can have a complete collagen structure and function when recombinantly expressed in vitro. However, its collagen expression level is not high, and it only contains the GER tripeptide at the C-terminus of the polypeptide, and the hydrophilic effect may be poor, and there is still room for improvement in obtaining complete collagen function. Summary of the invention

[0005] The present invention provides the following contents:

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

[0007] The technical solution adopted by 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 repetitions of the sequence shown in SEQ ID No. 1, wherein 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, each repeated sequence is directly connected.

[0010] 3. A 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 having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with 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 in 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, and the amino acid sequence retains the triple helical structure of the amino acid sequence of SEQ ID No. 2, wherein the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent 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, six histidine tags are added to its N-terminus, which are connected via the ENLYFQ (SEQ ID No. 4) sequence, which can be cut off by TEV protease to directly 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 also includes 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 genetically engineered Escherichia coli by screening.

[0029] The fermentation culture of the genetically engineered Escherichia coli in step 2) is as follows: a single colony of the genetically engineered Escherichia 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 amount, cultured at 37°C until OD600 is between 0.4-0.6, 0.5 mM IPTG is added to induce expression, the temperature is lowered to 16°C, and the culture is continued at 120rpm 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) enzymatically cleaving the recombinant human collagen using TEV protease.

[0032] The advantages of the recombinant collagen of the present invention are as follows: (1) The recombinant human collagen produced by the collagen sequence provided by the present invention is 100% identical to the corresponding part of the natural collagen gene sequence, has a triple helix structure, has good hydrophilicity and stability, strong cell adhesion activity, and high protein expression. (2) The E. coli expression system is adopted, with short culture time and low cost, which can be adapted to large-scale industrial production. (3) The N-terminus is connected to 6 histidine-specific affinity purification tags, and the obtained product has high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 :Prediction map of hydrophilicity and hydrophobicity 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 :Thermostability 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 present invention is clearly and completely described below in conjunction with specific embodiments.

[0039] Example 1 Gene design and expression

[0040] 1. Gene design and synthesis of human collagen C3-15

[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 a 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 the recombinant human collagen C3-15. The DNA sequence SEQ ID No. 3 of the recombinant human collagen C3-15 was submitted to GenScript Biotech Inc. for whole 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 min. Heat shock was performed in a 42°C water bath for 90 s and 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 h. The transformed cells were spread on an LB plate (containing 50 μg / ml kanamycin) and inverted in a 37°C incubator for overnight culture. 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 LB medium (containing 50 μg / ml kanamycin). The bacterial solution was inoculated into 2YT medium (16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride) at a 1% inoculum to expand the culture. The culture was cultured at 37°C until the OD600 was between 0.4 and 0.6. IPTG was added at 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 collected by centrifugation.

[0047] 4. Purification of recombinant human collagen.

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

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

[0050] 5. Protein concentration detection

[0051] The concentration of protein samples was detected by ultraviolet absorption method: the ultraviolet absorption of samples at 215nm and 225nm was measured respectively, and the protein concentration was calculated by the empirical formula C (μg / ml) = 144 × (A215-A225). Note that the detection should be performed when A215 < 1.5. The principle of this method is to determine the characteristic absorption of peptide bonds under far ultraviolet light. It is not affected by the content of chromophores, has few interfering substances, and is easy to operate. It is suitable for detecting human collagen and its analogs that are not colorable with Coomassie Brilliant Blue. Recombinant human collagen C3-15, 250ml of bacterial solution can obtain 750mg 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 tested by SDS-PAGE

[0056] Sample treatment: Add 15 μl of 4× loading buffer to 45 μl of sample, mix well, boil in boiling water for 10 min, and cool naturally for later use.

[0057] Protein electrophoresis: GenScript SurePAGETM precast gel (4-12%) was used for sample loading, and electrophoresis was performed at 140V for about 90 minutes until the bromophenol blue band reached the bottom of the gel. Protein staining instrument (GenScript eStain® L1) was used for staining and destaining. Due to the special amino acid sequence and structural characteristics of collagen, its migration speed is slow. The results are shown in 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 the purity was tested by SDS-PAGE after 1 week, 2 weeks, 3 weeks and 6 weeks. The results are as follows: 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. The circular dichroism of C3-15 recombinant humanized collagen was identified. The results are as follows: Figure 3 As shown in . The protein has a positive peak at 225nm, 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 detection 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 protein solution to be tested and the blank PBS solution control to the 96-well plate, and let 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 of OD492nm. According to 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, 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 a triple helix, and its basic repeating unit is shown in SEQ ID No. 1: GERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGER, which is a type III peptide segment of human collagen.

2. The recombinant human collagen according to claim 1, characterized in that: The recombinant human collagen is C3-15, and the amino acid sequence is shown in SEQ ID No.

2.

3. The recombinant human collagen according to claim 2, characterized in that: The DNA sequence of the recombinant human collagen C3-15 is shown in SEQ ID No.

3.

4. A recombinant vector comprising the DNA sequence according to claim 3. A host cell comprising the recombinant vector according to claim 4.

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

7. The method for producing recombinant human collagen according to claim 1, characterized in that: (1) Transforming Escherichia coli with the recombinant vector of claim 4 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.

8. The method for producing recombinant human collagen according to claim 7, characterized in that: The steps for constructing the genetically engineered Escherichia coli described in step (1) are as follows: a. 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; b. Use the expression vector pET-32a to clone the DNA fragment and transform it into BL21DE3 Escherichia coli to screen and obtain genetically engineered Escherichia coli.

9. The method for producing recombinant human collagen according to claim 7, 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, 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.

10. The method for producing recombinant human collagen according to claim 7, 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 by ultrasonication, 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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