CN117886923A - Recombinant humanized collagen and encoding gene and application thereof - Google Patents
Recombinant humanized collagen and encoding gene and application thereof Download PDFInfo
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- CN117886923A CN117886923A CN202410290161.2A CN202410290161A CN117886923A CN 117886923 A CN117886923 A CN 117886923A CN 202410290161 A CN202410290161 A CN 202410290161A CN 117886923 A CN117886923 A CN 117886923A
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- humanized collagen
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
The invention relates to recombinant humanized collagen and a coding gene and application thereof, belonging to the technical fields of genetic engineering and protein engineering. The invention is based on the amino acid sequence of human collagen, designs the amino acid sequence by means of bioinformatics and the like, realizes higher expression efficiency and high stability on the premise of maintaining high bioactivity of the recombinant collagen, designs and obtains a brand new collagen sequence containing 658 amino acids, has large protein molecular weight, and can better play the biological function of the human collagen. The nucleotide sequence of the recombinant collagen is optimized by the codon preference of escherichia coli, a high-expression recombinant strain is constructed, and the recombinant collagen produced by fermenting the recombinant strain has no exogenous amino acid such as any tag and the like, is recombinant humanized collagen, and has good safety. The invention also provides a low-cost and high-efficiency protein purification process which is suitable for large-scale production.
Description
Technical Field
The invention relates to recombinant humanized collagen and a coding gene and application thereof, belonging to the technical fields of genetic engineering and protein engineering.
Background
Collagen is an important structural protein, and is one of the most abundant proteins in many organisms. The main function of the collagen is to provide structural support for the body and maintain the stability and elasticity of the tissues. In tissues and organs, collagen is mainly present in tissues such as bones, joints, muscles, skin, blood vessels, cornea, and the like. In addition, collagen can be used as a signal molecule to regulate proliferation, differentiation and migration of cells, and has important effects on maintaining normal physiological functions and overall health of organisms. The recombinant collagen has the advantages of low toxicity, low antigenicity, low immunity, capability of guiding cell regeneration, good biocompatibility and the like, and is widely applied to industries such as biological medicine, cosmetics, food and the like.
In the present stage, large research institutions mainly carry out recombination work around human type III collagen. Some research institutions select a short segment of human collagen fragment as a repeating unit, and splice the segments together to express long-segment recombinant humanized collagen (such as CN102443057A, CN 106554410A), and the recombinant humanized collagen cannot replicate the sequence of the human collagen well and cannot fully exert the biological function of the human collagen; some research institutions can do some optimization work on the basis of the human collagen sequence, namely recombinant collagen (such as CN114262368A, CN 113735964A), but the recombinant collagen has low safety and is easy to cause adverse reactions such as allergy; in the prior art, purification tags are added on protein amino acid sequences to obtain products with high purity, and the purification cost of the scheme is high (such as CN 111004319A).
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a recombinant humanized collagen and a coding gene and application thereof. Experiments prove that the collagen produced by the invention has large molecular weight and can better exert the biological functions of the humanized collagen; no foreign amino acid such as any label, is recombinant humanized collagen, and has good safety; the preparation has better antioxidant activity and barrier repair biological activity; and the protein yield is high, the purification cost is low, and the method is more suitable for industrialized mass production.
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
a recombinant humanized collagen has an amino acid sequence shown in SEQ ID NO. 1.
A recombinant humanized collagen encoding gene, which encodes the recombinant humanized collagen.
According to the invention, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.
A recombinant expression vector containing the coding gene of the recombinant humanized collagen.
According to a preferred embodiment of the present invention, the plasmid vector of the recombinant expression vector is pET28a.
A recombinant expression strain containing the coding gene of the recombinant humanized collagen or the recombinant expression vector.
According to a preferred embodiment of the present invention, the host bacterium of the recombinant expression strain is E.coli.
Further preferably, the E.coli is E.coli BL21 (DE 3).
The recombinant expression strain is applied to the preparation of recombinant humanized collagen.
A method for preparing recombinant humanized collagen by utilizing the recombinant expression strain to perform high-density fermentation comprises the following steps: fermenting and culturing the recombinant expression strain to OD in a fermenter 600 After 79-81, recombinant humanized collagen was induced to be expressed.
A method for purifying recombinant humanized collagen, comprising the steps of: and centrifuging the fermentation liquor after the recombinant expression strain is induced and cultured to obtain bacterial mud, homogenizing bacterial liquor after bacterial mud is resuspended, removing impurity proteins, centrifuging, clarifying by a hollow fiber membrane, concentrating by an ultrafiltration membrane and performing ion exchange chromatography to obtain recombinant humanized collagen purified stock solution.
According to the invention, the impurity-removed protein is removed by an acid precipitation method, and the pH of the bacterial liquid is adjusted to 2.7-2.9.
According to a preferred embodiment of the invention, the hollow fiber membrane has a molecular weight cut-off of 250kDa.
According to a preferred embodiment of the invention, the ultrafiltration membrane has a molecular weight cut-off of 10kDa.
Preferably, according to the present invention, the ion exchange chromatography is anion exchange chromatography.
The application of the recombinant humanized collagen in preparing food, medicine, cosmetics, health care products or appliance products.
The beneficial effects are that:
the invention is based on the amino acid sequence of human collagen, designs the amino acid sequence by means of bioinformatics and the like, realizes higher expression efficiency and high stability on the premise of maintaining high bioactivity of the recombinant collagen, designs and obtains a brand new collagen sequence containing 658 amino acids, has large protein molecular weight, and can better play the biological function of the human collagen. The nucleotide sequence of the recombinant collagen is optimized by the codon preference of escherichia coli, a high-expression recombinant strain is constructed, and the recombinant collagen produced by fermenting the recombinant strain has no exogenous amino acid such as any tag and the like, is recombinant humanized collagen, and has good safety. The invention also provides a low-cost and high-efficiency protein purification process which is suitable for large-scale production. Compared with the collagen product sold in the market, the recombinant humanized collagen prepared by the technical scheme has better antioxidant activity and barrier repair capability, so that the recombinant humanized collagen has good practical application value.
Drawings
FIG. 1 is a recombinant bacteriumE. coliSDS-PAGE detection graph after BL21/pET28a-Col induced expression; wherein lane M represents a standard protein having a molecular weight of 180 kDa; lane 1 represents a control bacteriumE. coliBL21/pET28a cell-destroying supernatant; lane 2 represents recombinant bacteriaE. coliBL21/pET28a-Col cell supernatant.
FIG. 2 is a recombinant bacteriumE. coliPerforming SDS-PAGE protein electrophoresis of high-density culture induction phase of a 5-L fermentation tank by BL21/pET28 a-Col; wherein,lane M represents a standard protein with a molecular weight of 180 kDa; lane 1 represents pre-induction; lane 2 represents induction for 5h; lane 3 represents induction for 10h; lane 4 represents induction for 12h; lane 5 represents induction for 15h.
FIG. 3 is a bar graph of DPPH radical scavenging for different collagens; wherein, the comparative example represents a commercial recombinant collagen comparative experiment group; col represents the experimental group of recombinant humanized collagen prepared using example 5 of the present invention.
FIG. 4 is a bar graph showing the relative expression of FLG mRNA in HaCaT cells under the action of different collagens; wherein, the control group 1 represents a blank control group; control group 2 represents a commercially available recombinant collagen control group; col represents the recombinant humanized collagen Col set prepared in example 5 of the present invention; in the figure, P <0.01 (×0.0001 (×0) and P <0.0001 (×0) are considered to have significant differences compared to control group 1.
Detailed Description
The technical scheme of the present invention will be described in further detail with reference to examples and drawings, but the scope of the present invention is not limited thereto. The medicines and reagents related to the examples are common commercial products unless specified; the experimental procedures referred to in the examples, unless otherwise specified, are conventional in the art. The percentages referred to in the examples are mass percentages unless otherwise indicated.
Example 1: recombinant humanized collagen sequence design
The NCBI database is utilized to obtain the amino acid sequence (GenBank: KAI 2526099.1) of the natural human III-type collagen alpha 1 chain protein, the deep mining analysis of the sequence information is utilized, and the means such as bioinformatics and the like are utilized to carry out sequence design on the hydrophilicity and hydrophobicity, charge distribution and protein space structure of the amino acid, so that the recombinant collagen can realize higher expression efficiency and high stability on the premise of maintaining high bioactivity. The recombinant collagen of the invention has 658 amino acids in total, the specific amino acid sequence is shown as SEQ ID NO.1, the sequence contains the main bioactive site of the natural human III type collagen alpha 1 chain, the realization of the biological function of the recombinant collagen is ensured, the sequence does not contain any exogenous amino acid such as labels, and the recombinant collagen is recombinant humanized collagen, and has high biocompatibility and low use risk.
Example 2: recombinant bacteriumE. coliConstruction of BL21/pET28a-Col
The coding gene of the recombinant humanized collagen shown in SEQ ID NO.1 in example 1 is subjected to codon optimization according to the codon preference of the escherichia coli, and the nucleotide sequence of the optimized coding gene is shown as SEQ ID NO.2 and is named Col. The nucleotide sequence of Col entrusts the large gene to synthesize and clone to E.coli expression vector pET28a to transform toE. coliIn TOP10, recombinant expression vectors pET28a-Col are obtained on the premise of ensuring that reading frames are not shifted, and the recombinant expression vectors pET28a-Col are successfully constructed through DNA sequencing comparison.
The verified recombinant expression vector pET28a-Col is transformed into escherichia coli BL21 (DE 3) according to an escherichia coli operation manual, kanamycin antibiotics are utilized to screen recombinant expression transformants, and PCR verification is carried out to verify recombinant bacteria for expressing recombinant humanized collagenE. coliBL21/pET28a-Col construction was successful.
Example 3: recombinant bacteriumE. coliInduced expression of BL21/pET28a-Col
Picking recombinant bacteriaE. coliBL21/pET28a-Col single colony was cultured overnight at 37℃in LB liquid medium (1% NaCl,0.5% yeast extract, 1% peptone) containing 50. Mu.g/mL kanamycin, and then inoculated to TB medium (1.18% peptone, 2.36% yeast extract, 0.94% K) at an inoculum size of 2% after 200rpm 2 HPO 4 ，0.22% KH 2 PO 4 0.4% glycerol), at 37℃and 200rpm to OD 600 0.7, adding 0.5mM IPTG (isopropyl-beta-D-thiogalactoside) with final concentration for induced expression, culturing at 25deg.C for 16 hr, centrifuging to collect thallus, re-suspending with Tris buffer (20 mM Tris, pH 7.5), ultrasonic crushing, subjecting the supernatant and precipitate to SDS-PAGE, and detecting the recombinant humanized collagen with theoretical size of 60.08kDa, wherein SDS-PAGE result is shown in figure 1E. coliBL21/pET28a-Col cell supernatant (lane 2) and blank controlE. coliBL21/pET28a (lane 1) is in theoryThere is a distinct protein band (indicated by the arrow) at the size. The above results indicate that the recombinant humanized collagen of the present invention can be successfully induced to express.
Example 4: recombinant bacteriumE. coliBL21/pET28a-Col 5-L tank high-density culture
Recombinant bacteria are used for preparing the recombinant bacteriaE. coliBL21/pET28a-Col was subjected to 5-L tank high-density culture. The bacterial liquid in the glycerol tube was inoculated into 60 mL seed medium (1% peptone, 1% sodium chloride, 0.5% yeast powder) containing 50. Mu.g/mL kanamycin at an inoculum size of 1% by weight, and cultured at 37℃for about 15 hours at 180 rpm. The whole seed liquid after cultivation was transferred into a 5-L fermenter containing 3L fermentation medium (3% yeast powder, 1.5% glycerol, 3.25% ammonium sulfate, 0.2% sodium chloride, 1.0% disodium hydrogen phosphate dodecahydrate, 0.31% potassium dihydrogen phosphate, 0.17% magnesium sulfate heptahydrate, 0.02% defoamer, 0.43% trace element, pH adjusted to 7.0 with sodium hydroxide; 500mL trace element formulation: boric acid 1.625g, manganese sulfate monohydrate 0.275g, cupric chloride dihydrate 0.3325g, zinc sulfate monohydrate 0.1375g, cobalt chloride 0.211g, sodium molybdate 0.1735 g). The initial fermentation parameters were set as follows: stirring at 150rpm, fermenting at 37deg.C, ventilating at 4L/min, regulating dissolved oxygen, stirring to 20%, and automatically adding ammonia water to control pH to 7.0. Feeding is started after dissolved oxygen is raised to 35% (feed medium formula: 3% ammonium sulfate, 0.22% magnesium sulfate heptahydrate, 30% glycerol, 0.5% microelements), initial feeding speed is 0.8 mL/min, dissolved oxygen is regulated by increasing ventilation and maintained at 20%, and OD is reached 600 After 80 mM IPTG was added, the temperature was adjusted to 25℃and the dissolved oxygen was controlled at about 30% for 10-20h. Samples were taken at regular intervals after induction time and the fermentation broth was subjected to SDS-PAGE detection.
As shown in FIG. 2, the SDS-PAGE detection result shows that the yield of the recombinant humanized collagen in the fermentation broth gradually increases along with the induction time, and the gray values of the standard protein and the recombinant humanized collagen are determined by analysis of BioAnaly biological analysis software, so as to calculate the recombinant strainE. coliWhen BL21/pET28a-Col is subjected to 5-L tank high-density fermentation induction for 15h, the yield of the recombinant humanized collagen is 8.3 g/L, thereby realizing the high expression of the recombinant humanized collagenEfficiency is improved.
Example 5: purification preparation of recombinant humanized collagen
Recombinant bacteria are used for preparing the recombinant bacteriaE. coliPerforming high-density fermentation on BL21/pET28a-Col in a 5-L tank, centrifuging the fermentation liquor after induced culture for 15h at 8000rpm to obtain bacterial sludge, and purifying the bacterial sludge to obtain a recombinant humanized collagen pure product through the following purification steps:
(1) impurity removal protein: the pH of the bacterial liquid after bacterial mud re-suspension homogenization is adjusted to 2.8 by low-concentration hydrochloric acid, and the bacterial liquid is continuously stirred in the acid adding process, so that partial peracid in the process is prevented, and the recombinant humanized collagen is denatured and precipitated;
(2) and (3) centrifuging: centrifuging to remove acid precipitated impurity protein, and regulating pH of the supernatant to 7.0 with low concentration sodium hydroxide;
(3) clarifying by a hollow fiber membrane: filtering the centrifugal supernatant by a 250kDa hollow fiber membrane, sterilizing and removing macromolecular impurities;
(4) concentrating by ultrafiltration membrane: concentrating the feed liquid by using a 10kDa ultrafiltration membrane bag, and removing small molecular impurities;
(5) ion exchange chromatography: anion exchange chromatography is adopted, recombinant humanized collagen is positively charged, and the flow-through liquid is directly collected during chromatography sample loading, impurities such as negatively charged impurity proteins and endotoxin can be adsorbed on a column, so that the aim of removing the impurities is achieved, and the obtained flow-through liquid is the purified stock solution.
The purity of the protein purified by the steps is more than 97%, and the yield is more than 60%.
Example 6: antioxidant experiment of recombinant humanized collagen
An appropriate amount of the purified stock solution of recombinant humanized collagen of example 5 was placed in a test tube, 2.5mL of DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) free radical solution (0.1 mmoL/L) in 95% ethanol was added, the mixture was vigorously shaken for 10s, then left to react at room temperature for 30min, after the reaction was completed, the absorbance of the reaction mixture was measured at 517nm, distilled water was used as a blank control instead of the sample solution, and a comparative experiment was performed with commercially available recombinant collagen (comparative example, the recombinant protein was purchased from Hebei Nake biosciences, model 3CH-FD 05).
DPPH radicalScavenging activity= (OD Blank space -OD Sample of ）/OD Blank space ，
Wherein OD Blank space Absorbance value, OD, of the blank control group Sample of Absorbance values for recombinant humanized collagen or commercially available recombinant collagen groups.
As shown in FIG. 3, the recombinant humanized collagen Col of the present invention has an obvious DPPH radical scavenging ability compared with the blank, and the DPPH radical scavenging rate is higher than that of the commercially available recombinant collagen, so that the recombinant humanized collagen provided by the present invention has an obvious antioxidant effect.
Example 7: barrier repair experiments
Filaggrin (FLG) is one of the important components of the keratinized envelope, ensuring the integrity of the skin barrier. FLG deficiency can lead to disorder of lipid bilayer structure, delayed maturation, reduced cell tightness of the stratum corneum, enhanced intercellular permeability and reduced photoprotection, ultimately leading to impaired skin barrier.
Human immortalized keratinocytes (HaCaT cells, purchased from Shanghai cell Bank, national academy of sciences) were collected, and cell suspensions were prepared using high-sugar DMEM cell culture broth, 2mL of the cell suspension was added to each well of a 6-well plate, and the number of cells was 2.6X10 5 /well. A blank control group (control group 1), a commercially available recombinant collagen control group (control group 2), and a recombinant humanized collagen Col group (experimental group) prepared in example 5 of the present invention were experimentally set, and 3 duplicate wells were set in each group. The 6-well plate was placed in a cell incubator (37 ℃, 5% CO) 2 ) After 24h incubation, discarding the culture solution when the cell fusion rate reaches 50% -60%, and adding 2mL of high-sugar DMEM cell culture solution into each hole of the blank control group; the other two groups are respectively added with 2mL of high-sugar DMEM cell culture solution containing corresponding collagen, wherein the concentration of the collagen in the high-sugar DMEM cell culture solution is 1mg/mL. 6-well plates were placed in an incubator (37 ℃, 5% CO) 2 ) After incubation for 24h, cells were washed twice with 2mL of PBS buffer per well, and the cells were isolated according to Total RNA extraction kit (FastPure Cell/Tissue Total RNA Isolation Kit in NanjinopranV2 kit (RC 112-01)), adding 1mL RNAiso Plus, blowing to lyse cells, collecting sample, performing RNA extraction, reverse transcription and fluorescent quantitative PCR detection experiments according to the kit specification, detecting the relative expression level of mRNA of barrier-related protein FLG, and adopting 2 -△△CT The method performs the calculation.
As shown in FIG. 4, compared with the blank control group (control group 1), the mRNA expression capacity of FLG in HaCaT cells is promoted by different collagens, and compared with the blank control group (control group 2), the mRNA relative expression capacity of FLG can be obviously improved by the commercially available recombinant collagen (control group 2) and the recombinant humanized collagen Col (experimental group) prepared by the embodiment 5 of the invention, wherein the mRNA expression capacity of FLG is promoted by the prepared recombinant humanized collagen, which is stronger than that of the commercially available recombinant collagen, so that the recombinant humanized collagen has an obvious skin barrier repair function.
Claims (13)
1. The recombinant humanized collagen is characterized in that the amino acid sequence of the recombinant humanized collagen is shown as SEQ ID NO. 1.
2. A gene encoding recombinant humanized collagen, wherein the gene encodes the recombinant humanized collagen of claim 1.
3. The recombinant humanized collagen encoding gene according to claim 2, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 2.
4. A recombinant expression vector comprising the recombinant humanized collagen encoding gene according to claim 2.
5. The recombinant expression vector of claim 4, wherein the plasmid vector of the recombinant expression vector is pET28a.
6. A recombinant expression strain comprising the recombinant humanized collagen encoding gene of claim 2 or the recombinant expression vector of claim 4.
7. The recombinant expression strain of claim 6, wherein the host strain of the recombinant expression strain is escherichia coli.
8. The recombinant expression strain of claim 7, wherein the E.coli is E.coli BL21 (DE 3).
9. Use of the recombinant expression strain of claim 6 for the preparation of recombinant humanized collagen according to claim 1.
10. A method for preparing recombinant humanized collagen by high-density fermentation using the recombinant expression strain of claim 6, comprising the steps of: fermenting the recombinant expression strain of claim 6 to OD in a fermenter 600 After 79-81, recombinant humanized collagen was induced to be expressed.
11. A method for purifying recombinant humanized collagen, comprising the steps of: centrifuging the fermentation broth after the recombinant expression strain is induced and cultured to obtain bacterial sludge, homogenizing the bacterial liquid after bacterial sludge is resuspended, removing impurity proteins, centrifuging, clarifying by a hollow fiber membrane, concentrating by an ultrafiltration membrane, and performing ion exchange chromatography to obtain the recombinant humanized collagen purification stock solution.
12. The method of purifying recombinant humanized collagen according to claim 11, wherein one or more of the following conditions are satisfied:
i. the impurity-removing protein is removed by adopting an acid precipitation method, and the pH value of the bacterial liquid is adjusted to 2.7-2.9;
the molecular weight cut-off of the hollow fiber membrane is 250kDa;
the ultrafiltration membrane has a molecular weight cut-off of 10kDa;
the ion exchange chromatography is anion exchange chromatography.
13. The use of the recombinant humanized collagen according to claim 1 for the preparation of food, pharmaceutical, cosmetic, health or appliance products.
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