Recombinant collagen based on engineering bacterium expression and preparation method thereof

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to recombinant collagen based on engineering bacterium expression and a preparation method thereof, and the recombinant collagen is formed by connecting repetitive units derived from human III type collagen in series for three times; the invention also discloses a base sequence of the coding gene of the recombinant collagen, efficient expression and accurate folding of target protein are realized through genetic modification of engineering bacteria, a light-operated dynamic folding process and an orthogonal purification strategy, and cascade purification of a His6-SUMO tag and an intein-CBD module is utilized to obtain the recombinant collagen. The product purity and the native conformation retention rate are remarkably improved, and the technical problems that hydroxyproline depends on exogenous addition, the folding controllability is poor and the purification efficiency is low in a traditional process are solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant collagen protein expressed by an engineered bacterium and a preparation method thereof. Background Art

[0002] Collagen is the main component of the human extracellular matrix. Its unique triple helix structure is composed of repeated Gly-XY sequences (X and Y are usually proline and hydroxyproline), which gives it high mechanical strength and biological activity. It is widely used in tissue repair, drug carriers and medical beauty fields. However, due to the lack of endogenous prolyl hydroxylase (P4H) in the prokaryotic expression system, the hydroxyproline content is less than 50% of that in natural collagen, which seriously weakens the stability of the triple-helix hydrogen bond network. Although the eukaryotic system has partial hydroxylation ability, its secretion efficiency is low and high-density fermentation easily leads to endotoxin accumulation, which makes it difficult to meet the needs of large-scale production. At the same time, the existing engineered bacteria lack the ability to dynamically regulate post-translational modifications. The fluctuation of the supply of exogenously added vitamin C leads to uneven hydroxylation sites, and the activity of hydroxylase is limited by the instability of the intracellular redox microenvironment. In addition, long tandem repeat genes are prone to induce ribosome arrest and mRNA secondary structure formation, resulting in an increased translation error rate. The misfolded products aggregate in the cell to form inclusion bodies, and the reliance on in vitro renaturation processes further increases costs and complexity. The production of recombinant collagen faces multiple technical challenges. In terms of purification process, traditional methods mostly rely on metal chelation chromatography or protease cleavage, which carries the risk of metal ion residues and cleavage sites destroying the natural structure. The emerging self-assembly purification technology may change the collagen conformation due to the introduction of exogenous carriers, limiting its medical-grade application. Although recent studies have attempted to optimize expression and folding through gene tandem duplication, co-expression of folding auxiliary molecules or light-controlled induction systems, there are still significant limitations: gene duplication design does not solve the frameshift mutation caused by ribosome sliding, static molecular chaperone co-expression cannot adapt to dynamic folding requirements, and the light-controlled system lacks penetration to regulate deep bacterial communities in high-density fermentation.

[0003] Therefore, there is an urgent need to develop a new strategy that can synergistically achieve efficient expression, precise modification and structural biomimetic of recombinant collagen. Summary of the Invention

[0004] In response to the above situation, the present invention provides a recombinant collagen protein expressed based on engineered bacteria and a preparation method thereof, which utilizes a light-responsive promoter to regulate the dual-signal peptide secretion pathway, combines the hydroxyproline in situ synthesis module with the dynamic regulation of the oxidative folding microenvironment, and significantly improves the triple helix hydrogen bond density and thermal stability; at the same time, a self-cleaving tag and amyloid fiber self-assembly purification system are designed to achieve a breakthrough in the high-density expression, precise folding, and structural bionic integration of recombinant collagen protein.

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

[0006] The present invention provides a recombinant collagen protein expressed by an engineered bacterium, wherein the recombinant collagen protein is composed of a repeating unit repeated three times in series, wherein the repeating unit is selected from a continuous segment containing 252 amino acid residues in human type III collagen.

[0007] Furthermore, the amino acid sequence of the recombinant collagen is shown as SEQ ID No.1.

[0008] Furthermore, a flexible peptide segment is inserted into the tandem portion of the recombinant collagen to obtain recombinant collagen I, and the amino acid sequence of the recombinant collagen I is shown in SEQ ID No. 2.

[0009] Furthermore, the amino acid sequence of the flexible peptide segment is GPGER.

[0010] Furthermore, the amino acid sequence of the repeating unit of the recombinant collagen is shown as SEQ ID No.3.

[0011] Furthermore, the amino acid sequence of the human type III collagen is shown in SEQ ID No.4.

[0012] Furthermore, the base sequence of the gene encoding the recombinant collagen is shown as SEQ ID No.5.

[0013] Furthermore, the base sequence of the gene encoding the recombinant collagen I is shown in SEQ ID No.6.

[0014] Furthermore, the recombinant collagen and recombinant collagen I are expressed by genetically modified engineering bacteria, and the engineering bacteria are selected from one of BL21 (DE3) competent cells or Pichia pastoris GS115.

[0015] Preferably, the genetically modified bacteria are constructed from BL21 (DE3) competent cells.

[0016] The present invention also provides a method for preparing recombinant collagen based on expression of engineered bacteria, which specifically comprises the following steps:

[0017] Step 1: Design recombinant collagen (amino acid sequence SEQ ID No. 1), fuse the His6-SUMO double tag and TEV restriction site and intein-chitin binding domain (CBD), encode and optimize the gene, clone it into the pUC57 vector, and then transform it into the pET-28a vector after double enzyme digestion to obtain the recombinant plasmid;

[0018] Step 2: The recombinant plasmid and the pGro7 molecular chaperone plasmid were co-transformed into genetically modified engineering bacteria, and double antibodies (kanamycin 50 μg / mL + chloramphenicol 34 μg / mL) were added to screen for positive bacteria. The transformation process of the genetically modified engineering bacteria was as follows: the P4H hydroxylase gene and the GULO gene (vitamin C synthesis gene) from the archaea were concatenated and inserted into the pKO3 vector containing the attB recombination site, and the integration plasmid was constructed using CRISPR-Cas9. The integration plasmid was transformed into BL21 (DE3) competent cells, and the site-specific integration was achieved by the arabinose-inducible λ-Red system. The endogenous trxB gene was knocked out and the light-controlled LOV2-TrxB fusion protein was introduced and inserted into the genomic attTn7 site to obtain the genetically modified engineering bacteria;

[0019] Step 3: Prepare the positive bacteria into a seed liquid, transfer the seed liquid to TB medium (containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 5mM MgSO4) at a 1% inoculum volume, and culture at 37°C and 200rpm to OD600 = 0.6, then cool to 20°C, add 0.1mM IPTG (isopropylthiogalactoside) and 1% arabinose, supplement with 5mM reduced glutathione and 2mM α-ketoglutarate, and monitor the proline concentration in real time by ProSense fluorescent probe, dynamically feed and maintain it at 8-10mM, and use 650nm near-infrared light for dynamic regulation and 470nm blue light to activate the promoter and optimize the oxidative folding system to obtain the target bacteria;

[0020] Step 4: The target bacteria are treated by periodic temperature-dissolved oxygen oscillation fermentation. After the fermentation is completed, the precipitate is collected by centrifugation, the precipitate is ultrasonically broken in an ice bath, and the supernatant is centrifuged to obtain the crude protein.

[0021] Step 5: The supernatant was passed through a Ni-NTA column to capture the His6-tagged protein, dialyzed, and then SUMO protease cleaved. 50 mM DTT was added to activate the intein self-cleavage, and the residual CBD tag was removed by passing through a chitin column. After chromatography, ultrafiltration concentration, and freeze-drying, recombinant collagen was obtained.

[0022] The beneficial effects achieved by the present invention are as follows:

[0023] The present invention provides a recombinant collagen protein expressed by engineered bacteria. Based on the collaborative innovation of gene design and preparation technology, an efficient and controllable recombinant collagen protein production system is constructed, which solves the core problems of traditional technologies such as the dependence of hydroxyproline synthesis on exogenous precursors, uncontrollable folding pathways and cumbersome purification processes. At the sequence design level, the continuity and stability of the triple helix structure were significantly enhanced by precisely concatenating functional fragments of human type III collagen. At the same time, a preferred codon optimization strategy for engineered bacteria was adopted to introduce gene modules of the archaeal P4H hydroxylase and vitamin C self-synthesis pathway. The constructed genetically modified engineered bacteria can independently complete the synthesis and incorporation of hydroxyproline, completely get rid of dependence on exogenous additions, and reproduce the hydrogen bond network characteristics of natural collagen at the molecular level. In terms of preparation technology, a multi-wavelength light-controlled dynamic regulation system was developed. Through the synergistic effect of 650nm near-infrared light and 470nm blue light, the temporal and spatial precise matching of the target protein expression intensity and the intracellular oxidative folding microenvironment was achieved, replacing traditional chemical inducers and static redox regulation methods, ensuring the efficient assembly of the triple helix structure and the directional formation of disulfide bonds. Further combined with the orthogonal purification strategy, the cascade reaction characteristics of the His6-SUMO tag and the Intein-CBD system were utilized to greatly improve the purity and natural conformation retention rate of the target product. In addition, the introduction of temperature-dissolved oxygen oscillation fermentation technology dynamically balances folding dynamics and oxidative stress response by periodically switching culture conditions, thereby solving the common industry challenges of energy barriers and incorrect aggregation in complex protein folding pathways, and providing a new paradigm for the large-scale production of highly uniform collagen materials.

[0024] The present invention achieves the controllable synthesis and structural biomimetic of recombinant collagen through the gene design-process regulation-purification strategy, showing significant advantages in avoiding exogenous contamination, reducing production costs and improving product stability, and provides a new direction for the production and application of protein-based biomaterials based on engineered bacteria expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the plasmid map of the recombinant collagen in Example 1;

[0026] Figure 2 This is a simulated diagram of the tertiary structure of the recombinant collagen in Example 1;

[0027] Figure 3 The results of the GC base content changes before and after the codon optimization of the recombinant collagen gene encoding Example 1 are shown;

[0028] Figure 4 This is the Ramachandran plot of the recombinant collagen in Example 1;

[0029] Figure 5 This is a simulated diagram of the tertiary structure of recombinant collagen I in Example 2;

[0030] Figure 6 The results of the GC base content changes before and after the codon optimization of the recombinant collagen I gene encoding Example 2 are shown;

[0031] Figure 7 This is the Ramachandran plot of recombinant collagen I in Example 2;

[0032] Figure 8 This is the SDS-PAGE gel electrophoresis test result of recombinant collagen I in Example 2;

[0033] Figure 9 The results of the cell adhesion experiment of recombinant collagen in Example 1 are as follows;

[0034] Figure 10 This is the rat skin wound repair result of recombinant collagen I in Example 2. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0037] In the following examples, unless otherwise specified, conventional methods are used; the materials used in the following examples, unless otherwise specified, are all new materials purchased from the market.

[0038] Example 1: This example provides a recombinant collagen protein expressed by an engineered bacterium, the amino acid sequence of the recombinant collagen protein is shown in SEQ ID No. 1.

[0039] This embodiment also provides a method for preparing recombinant collagen based on expression of engineered bacteria, which specifically comprises the following steps:

[0040] Step 1: Gene design and recombinant plasmid construction

[0041] A continuous segment of 252 amino acids derived from human type III collagen was used as a repeating unit. The repeating unit was concatenated three times using GeneOptimizer software to obtain recombinant collagen (amino acid sequence: SEQ ID No. 1). A His6-SUMO double tag was fused to the N-terminus of the recombinant collagen, and a TEV restriction site and Intein-CBD were added to the C-terminus to obtain the recombinant collagen to be encoded. The recombinant collagen to be encoded was gene-edited and codon-optimized for Escherichia coli preference. The codons for glycine (Gly), proline (Pro), and arginine (Arg) were optimized (Gly→GGC, Pro→CCG, Arg→CGT) to obtain the encoding gene. NdeI and XhoI restriction sites were introduced at both ends of the encoding gene to obtain the full gene sequence. The full gene sequence was synthesized by GenScript and cloned into the pUC57 vector. The correctness was verified by Sanger sequencing, and the repeat sequence identity was 99.8%. The recombinant collagen was then transformed into the pET-28a vector containing PhyB-PIF6 by double enzyme digestion to obtain a recombinant plasmid.

[0042] Step 2: Engineering bacterial genome modification

[0043] Before introducing the recombinant plasmid into BL21(DE3) competent cells, the BL21(DE3) competent cells were genome-modified: the P4H hydroxylase gene and the GULO gene from archaea were concatenated and inserted into the pKO3 vector containing the attB recombination site. CRISPR-Cas9 was used to construct an integration plasmid, which was transformed into BL21(DE3) competent cells. Site-specific integration was achieved using the arabinose-inducible λ-Red system, the endogenous trxB gene was knocked out, and the light-controlled LOV2-TrxB fusion protein was introduced and inserted into the genomic attTn7 site to obtain a modified engineered bacterium. The recombinant pET-28a plasmid and the pGro7 molecular chaperone plasmid were co-transformed into the modified engineered bacterium, and double antibodies were added and screened to obtain positive bacteria.

[0044] Step 3: Light-controlled fermentation and dynamic folding

[0045] The positive bacteria were inoculated into 5 mL of LB medium containing double antibodies, cultured at 37 ° C and 200 rpm for 12 h to prepare seed liquid, and the seed liquid was transferred to 500 mL of TB medium (containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 5 mM MgSO4) at a 1% inoculum volume, cultured at 37 ° C and 200 rpm to OD600 = 0.6, then cooled to 20 ° C, 0.1 mM IPTG and 1% arabinose were added, 5 mM reduced glutathione and 2 mM α-ketoglutaric acid were supplemented, and the proline concentration was monitored in real time by the ProSense fluorescent probe, and dynamic feeding was maintained at 8-10 mM. At the same time, 650 nm near-infrared light (dynamically adjusted light intensity range of 0.1-10 mW / cm 2 ) and 470nm blue light (5mW / cm 2 ) activated the promoter and optimized the oxidative folding system, the intracellular oxidation potential (Eh) increased from -225mV to -181mV, and the recombinant collagen was continuously induced to obtain the target bacteria;

[0046] Step 4: Temperature control optimization and crude protein collection

[0047] The target bacteria were subjected to a periodic temperature-dissolved oxygen oscillation fermentation treatment, switching between two fermentation conditions every 2 hours: 28°C / 30% DO (dissolved oxygen level) and 37°C / 60% DO. After the target bacteria were fermented for 20 hours, they were centrifuged at 4°C and 6000g for 15 minutes, and the precipitate was collected. The precipitate was ultrasonically disrupted in an ice bath (20mM Tris-HCl pH 8.0, 500mM NaCl, 1% TritonX-114), and the supernatant was centrifuged to obtain the crude protein.

[0048] Step 5: Orthogonal purification and detection

[0049] The supernatant was passed through a Ni-NTA column to capture the His6-tagged protein (the eluate contained 250 mM imidazole). After dialysis, the N-terminal tag was removed by SUMO protease, and 50 mM DTT was added to activate intein self-cleavage, releasing the target protein. The remaining CBD tag was removed by passing it through a chitin column. The recombinant collagen was purified by SP Sepharose FF cation exchange chromatography (pH 5.0 acetate buffer, 0-1 M NaCl gradient elution), concentrated by ultrafiltration, and freeze-dried. The purity of the recombinant collagen was 99.4% as determined by HPLC and SDS-PAGE gel electrophoresis, and the endotoxin content was <0.03 EU / mg as confirmed by the LAL reagent method.

[0050] Example 2: This example provides a recombinant collagen I having a three-repeat tandem sequence and a flexible peptide segment. The amino acid sequence is shown in SEQ ID No. 2. The specific preparation method is as follows:

[0051] Step 1: Gene design and recombinant plasmid construction

[0052] A 252-amino acid fragment of human type III collagen (SEQ ID No. 3) was used as a repeating unit and concatenated three times using GeneOptimizer software. The flexible peptide GPGER was inserted between each repeating unit to obtain recombinant collagen I (amino acid sequence: SEQ ID No. 2). A His6-SUMO double tag was fused to the N-terminus, and a TEV restriction site and Intein-CBD were added to the C-terminus. The optimized coding gene (SEQ ID No. 6) was generated through Escherichia coli-preferred codon optimization and the codon base sequence of the flexible peptide gene, GGCCCGGGCGAACGT. NdeI / XhoI restriction sites were introduced at both ends of the gene to obtain the full gene sequence, which was synthetically cloned into the pUC57 vector and, after sequencing verification, subcloned into the pET-28a vector containing the PhyB-PIF6 light-controlled promoter to construct a recombinant expression plasmid.

[0053] Step 2: Engineering bacterial genome modification

[0054] Before introducing the recombinant plasmid into BL21(DE3) competent cells, the BL21(DE3) competent cells were genome-modified: the P4H hydroxylase gene and the GULO gene from archaea were concatenated and inserted into the pKO3 vector containing the attB recombination site. CRISPR-Cas9 was used to construct an integration plasmid, which was transformed into BL21(DE3) competent cells. Site-specific integration was achieved using the arabinose-inducible λ-Red system, the endogenous trxB gene was knocked out, and the light-controlled LOV2-TrxB fusion protein was introduced and inserted into the genomic attTn7 site to obtain a modified engineered bacterium. The recombinant pET-28a plasmid and the pGro7 molecular chaperone plasmid were co-transformed into the modified engineered bacterium, and double antibodies were added and screened to obtain positive bacteria.

[0055] Step 3: Light-controlled fermentation and dynamic folding

[0056] The positive bacteria were inoculated into 5 mL of LB medium containing double antibodies, cultured at 37 ° C and 200 rpm for 12 h to prepare seed liquid, and the seed liquid was transferred to 500 mL of TB medium (containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 5 mM MgSO4) at a 1% inoculum volume, cultured at 37 ° C and 200 rpm to OD600 = 0.6, then cooled to 20 ° C, 0.1 mM IPTG and 1% arabinose were added, 5 mM reduced glutathione and 2 mM α-ketoglutaric acid were supplemented, and the proline concentration was monitored in real time by the ProSense fluorescent probe, and dynamic feeding was maintained at 8-10 mM. At the same time, 650 nm near-infrared light (dynamically adjusted light intensity range of 0.1-10 mW / cm 2) and 470nm blue light (5mW / cm 2 ) activated the promoter and optimized the oxidative folding system, the intracellular oxidation potential (Eh) increased from -232mV to -169mV, and the expression of recombinant collagen was continuously induced to obtain the target bacteria;

[0057] Step 4: Temperature control optimization and crude protein collection

[0058] The target bacteria were subjected to periodic temperature-dissolved oxygen oscillation fermentation, switching between fermentation conditions of 28°C / 30% DO (dissolved oxygen level) and 37°C / 60% DO every 2 hours. After the target bacteria were fermented for 20 hours, they were centrifuged at 4°C and 6000g for 15 minutes, and the precipitate was collected. The precipitate was ultrasonically disrupted in an ice bath (20mM Tris-HCl pH 8.0, 500mM NaCl, 1% TritonX-114), and the supernatant was centrifuged to obtain the crude protein.

[0059] Step 5: Orthogonal purification and detection

[0060] The supernatant was passed through a Ni-NTA column to capture the His6-tagged protein (the eluate contained 250 mM imidazole). After dialysis, the N-terminal tag was removed by SUMO protease, and 50 mM DTT was added to activate intein self-cleavage, releasing the target protein. The remaining CBD tag was removed by passing the protein through a chitin column. Recombinant collagen I was obtained after purification by SP Sepharose FF cation exchange chromatography (pH 5.0 acetate buffer, 0-1 M NaCl gradient elution), ultrafiltration concentration, and freeze-drying. The purity of the recombinant collagen was 99.2% as determined by HPLC and SDS-PAGE gel electrophoresis, and the endotoxin content was <0.03 EU / mg as confirmed by the LAL reagent method.

[0061] Physical and chemical property analysis

[0062] The molecular weight, hydrophilicity, thermal stability and isoelectric point of the recombinant collagen and recombinant collagen I obtained in Example 1-2 were analyzed using protein bioanalysis software. The results are shown in Table 1.

[0063] The plasmid used to prepare recombinant collagen in Example 1 is as follows Figure 1 As shown, the three-dimensional structure of recombinant collagen is as follows Figure 2 As shown in the figure, the GC content changes of the base sequence encoding the recombinant collagen before and after optimization are shown in the figure. Figure 3 As shown, the Laplace plot of recombinant collagen is as follows Figure 4 shown.

[0064] The three-dimensional structure of recombinant collagen I prepared in Example 2 is shown in Figure 5 The results of the GC content changes before and after the optimization of the base sequence encoding recombinant collagen I are shown in Figure 6, Laplace plot of recombinant collagen I Figure 7 The results of SDS-PAGE gel electrophoresis are shown in Figure 8 .

[0065] Cell adhesion assay

[0066] The recombinant collagen prepared in Example 1 was dissolved in PBS and prepared into three concentrations: 1 mg / mL, 5 mg / mL, and 10 mg / mL, which were set as the low concentration group, the medium concentration group, and the high concentration group, respectively. 100 μL of the recombinant collagen solution was added to each well of a 96-well culture plate. The wells added with PBS were used as the control group. The plates were coated overnight at 4°C. The recombinant collagen solution in each well was replaced with PBS buffer containing 2% BSA and incubated at room temperature for 1 hour. The cell concentration of the skin fibroblast suspension was adjusted to approximately 5×10 4 cells / mL, wash the culture plate, remove the BSA solution, add 100 μL of cell suspension to each well and incubate for 2 hours, gently wash the plate with PBS to remove unattached cells, fix the cells with 4% formaldehyde solution for 15 minutes, stain with 0.1% crystal violet for 20 minutes to mark the attached cells, wash away the excess dye, add an appropriate amount of deionized water, and use a microplate reader to measure the absorbance of each well at OD492nm. The results are shown in Figure 9 .

[0067] Wound repair experiment

[0068] The recombinant collagen prepared in Example 1 was prepared into three dosage concentrations of 0.1%, 0.5%, and 1%, which were marked as low concentration group, medium concentration group, and high concentration group, respectively. Recombinant human epidermal growth factor gel was used as the positive control group. 60 healthy adult SD rats weighing 200-250g were randomly divided into 5 groups, with 12 rats in each group: Model group: only skin burns were caused, no therapeutic drugs were given, and an equal amount of normal saline was applied subsequently; Positive control group: after skin burns, recombinant human epidermal growth factor gel was applied; Low, medium, and high concentration groups: after skin burns, recombinant collagen of corresponding concentrations was applied. Burn model establishment and drug administration: SD rats were anesthetized with 4% chloral hydrate (10mL / kg) by intraperitoneal injection, and the back hair was shaved, with an area of about 3×3cm. 2, use a burn and scald instrument to heat the metal probe to 100℃, and press it vertically on the rat's back skin for 5 seconds, causing a third-degree burn wound with a diameter of about 2 cm, and the wound depth reaches the subcutaneous tissue. Drug administration began immediately after modeling, and was administered once a day at regular intervals. The positive control group applied recombinant human epidermal growth factor gel according to the recommended dose in the instructions, and the thickness of the drug application remained consistent. The mental state, diet, and wound healing of the rats were observed every day, and the wound healing time was recorded. The healing standard was complete epithelialization of the wound without exudation or redness and swelling; Wound area measurement: On the 1st, 3rd, 7th, 14th, and 21st days after modeling, the wound was covered with a transparent film, and the wound edge was traced. The wound area was calculated using image analysis software, and the results are shown in the figure. Figure 10 .

[0069] Table 1 Physicochemical properties of recombinant collagen and recombinant collagen I

[0070] Protein type Molecular weight (kDa) hydrophilicity Thermal stability Isoelectric point Recombinant collagen 67.68 -1.015 20.41 9.842 Recombinant collagen I 68.68 -1.029 21.03 9.841

[0071] The results in Table 1 show that the recombinant collagen and recombinant collagen I have similar physical and chemical properties, are highly stable, and are hydrophilic and soluble.

[0072] Figure 1 The restriction endonuclease sites are optimized in the designed recombinant collagen protein particles, with small errors in the cutting points, which is conducive to protein expression and purification.

[0073] Figure 2 The tertiary structure of recombinant collagen showed a tendency to aggregate, compared with Figure 5 The tertiary structure of recombinant collagen I has fewer linear regions and more folded conformations.

[0074] Figure 3 The GC content of the base sequence encoding the recombinant collagen gene was 61.34% before optimization and 71.03% after optimization, which adapted to the codon preference of the engineering bacteria.

[0075] Figure 4 The results of the Ramachandran plot of recombinant collagen showed that most of the amino acid residue sites fell in the allowed region, with glycine and proline sites being more concentrated. Figure 7 The Ramachandran plot of recombinant collagen I showed that the structure was better than that of recombinant collagen and the conformation was reasonable.

[0076] Figure 6 The GC content of the base sequence encoding the recombinant collagen I gene was 81.68% before optimization and 70.67% after optimization.

[0077] Figure 8 Gel electrophoresis results showed that the prepared recombinant collagen I had high purity and the molecular weight was in line with the expected design.

[0078] Figure 9The results of the cell adhesion experiment showed that compared with the control group, the cell proliferation rates of the three concentration experimental groups had significant differences, which could promote good cell growth and adhesion.

[0079] Figure 10 The results of the rat skin wound repair experiment showed that the skin repair effects of the medium and high concentration groups were similar to those of the positive control group, indicating that the recombinant collagen prepared in Example 1 can be well absorbed and utilized by the skin and has certain advantages as a skin care or repair material.

[0080] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

[0081] The present invention and its embodiments are described above. Such description is not restrictive. The drawings show only one embodiment of the present invention, and actual applications are not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the present invention, any method and embodiment similar to the technical solution without creative design shall fall within the scope of protection of the present invention.

Claims

1. A recombinant collagen protein expressed by an engineered bacterium, characterized in that: The recombinant collagen is composed of a repeating unit repeated three times in series, and the amino acid sequence is shown in SEQ ID No. 1; The repeating unit is selected from a continuous segment containing 252 amino acid residues in human type III collagen, and the amino acid sequence is shown in SEQ ID No. 3; The recombinant collagen is inserted into the tandem portion of the recombinant collagen to obtain recombinant collagen I, the amino acid sequence of which is shown in SEQ ID No. 2; The amino acid sequence of the flexible peptide segment is GPGER.

2. The recombinant collagen expressed by engineered bacteria according to claim 1, characterized in that: The base sequence of the gene encoding the recombinant collagen is shown in SEQ ID No. 5; The base sequence of the gene encoding the recombinant collagen I is shown in SEQ ID No.

6.

3. The recombinant collagen expressed by engineered bacteria according to claim 2, characterized in that: The recombinant collagen and recombinant collagen I are expressed using genetically modified BL21 (DE3) competent cells.

4. A method for preparing recombinant collagen expressed by engineered bacteria according to any one of claims 1 to 3, characterized in that: The specific steps include: Step 1: Design a recombinant collagen amino acid sequence with three tandem repeat units and construct a recombinant plasmid; Step 2: The recombinant plasmid and the pGro7 molecular chaperone plasmid were co-transformed into genetically modified BL21 (DE3) competent cells, and double antibody screening was added to obtain positive bacteria; Step 3: Cultivate positive bacteria and perform dynamic feeding and light regulation to obtain target bacteria; Step 4: treating the target bacteria with periodic temperature-dissolved oxygen oscillation fermentation, collecting the precipitate by centrifugation, crushing the precipitate by centrifugation, and obtaining a supernatant; Step 5: Capture and purify the protein from the supernatant to obtain recombinant collagen.

5. The method for preparing recombinant collagen based on expression of engineered bacteria according to claim 4, characterized in that: In step 2, the transformation process of the genetically modified BL21 (DE3) competent cells is as follows: the P4H hydroxylase gene and the GULO gene are concatenated and inserted into the pKO3 vector, an integration plasmid is constructed and transformed into BL21 (DE3) competent cells, the endogenous trxB gene is knocked out, and the LOV2-TrxB fusion protein is introduced to obtain genetically modified BL21 (DE3) competent cells.

6. The method for preparing recombinant collagen based on expression of engineered bacteria according to claim 4, characterized in that: In step 1, the recombinant plasmid is fused with a His6-SUMO double tag, a TEV restriction site, and an Intein-CBD, and the vector of the recombinant plasmid is pET-28a; In step 3, the standard of the dynamic feeding is to maintain the proline concentration at 8-10 mM, and the light regulation is performed using near-infrared light and 470 nm blue light.

Images