A method for biosynthesis of a blue copper peptide

Abstract

The application discloses a biosynthesis method of blue copper peptide, comprising the following steps: synthesizing a recombinant protein according to an amino acid sequence shown in SEQ ID NO. 1, synthesizing a gene coding the recombinant protein, constructing a recombinant plasmid by using the gene, transferring the recombinant plasmid into a host bacterium, carrying out fermentation culture to induce protein expression, separating the protein, carrying out enzyme cutting and purification, and finally carrying out complexing with copper ions to obtain the blue copper peptide. Compared with the prior art, the biosynthesis method of the blue copper peptide has the advantages of low production cost, green environmental protection and high yield by optimizing the design of the fusion protein, and the yield of the final product can be significantly improved, and the fusion protein has better structure and isoelectric point characteristics.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for the biosynthesis of blue copper peptide. Background Technology

[0002] Blue copper peptide (GHK-Cu) is a compound formed by the complexation of copper ions and GHK tripeptide. Its composition is glycyl-L-histidy1-L-lysine-copper. Initially isolated from human plasma in 1973, it is medically known for improving blood circulation, promoting the synthesis of nerve and neurotrophic factors, and possessing antioxidant, anti-inflammatory, and anti-cancer properties. In 1999, researchers suggested that copper peptides and their copper repair derivatives could act as activators for tissue remodeling and also as signaling peptides. Currently, it is mainly used in skin care products to promote the regeneration of skin epithelial tissue, restore youthful skin, reduce fine and deep wrinkles and scars, improve skin elasticity, promote the proliferation of keratinocytes and fibroblasts, thicken subcutaneous tissue, reduce skin fragility and sensitivity, increase skin elasticity and resilience, and maximize the absorption and utilization of skincare products on the face.

[0003] The preparation of copper peptides typically requires the prior synthesis or preparation of GHK tripeptides, which, as bioactive peptides, are widely used in experimental and clinical applications. Most are produced through chemical synthesis, but small-scale production of short peptides is extremely costly, and its development is mainly limited by drawbacks such as excessive reaction byproducts, environmental unfriendliness, low yield, and high prices of reaction substrates and reagents. Genetic engineering technology has successfully produced many proteins. Currently, the main steps in producing proteins through genetic engineering are isolating the target gene, constructing an expression vector, and producing the protein in *E. coli*. This is a highly efficient method for producing proteins with relatively large molecular weights, but its application is limited by the low expression levels of short peptides. For example, Chinese patent application 202011639055.9 discloses a high-purity preparation method for the recombinant copper peptide precursor—GHK tripeptide. However, the fusion protein used has a high isoelectric point and an excessively high repetition rate of the three substrate amino acids, resulting in very low protein expression levels. The GHK tripeptide yield is only 17.56 mg / L, lacking industrial application value. Summary of the Invention

[0004] Purpose of the invention: In order to solve the above problems, the purpose of this invention is to provide a method for the biosynthesis of copper peptide, which has high yield, expression level up to 3.5 g / L, and strong applicability.

[0005] Technical solution: The objective of this invention can be achieved through the following technical solution:

[0006] A method for biosynthesizing a blue copper peptide includes synthesizing a gene encoding the recombinant protein according to the amino acid sequence shown in SEQ ID NO.1, constructing a recombinant plasmid using the protein, then transferring the plasmid into a host bacterium, inducing protein expression through fermentation, separating the protein, purifying it by enzymatic digestion, and finally complexing it with copper ions to obtain the blue copper peptide.

[0007] The nucleotide sequence of the gene is shown in SEQ ID NO.2, or other nucleotide sequences encoding the same protein sequence obtained based on codon degeneracy.

[0008] Preferably, the plasmid is selected from pET28a, pET30a or pETDuat-1.

[0009] Preferably, the host bacterium is selected from Escherichia coli BL21(DE3) or JM109.

[0010] Preferably, the fermentation culture induces protein expression by using IPTG as the inducer at a concentration of 0.3-0.5 mM, and the induction conditions are 28-40℃ and 250-350 rpm for 4-7 h.

[0011] Preferably, the method for separating the protein includes: centrifuging to collect bacterial cells, disrupting the cell wall, centrifuging to collect inclusion body proteins, washing the inclusion body proteins, and dissolving the inclusion body proteins with a buffer solution.

[0012] Preferably, the enzyme digestion is performed using trypsin at a pH of 8.0-10.0 at 15-35°C for 4-8 hours. Most preferably, the enzyme digestion is performed at a pH of 9.0 at 25°C for 6 hours.

[0013] Preferably, the purification method employs ion exchange column chromatography; more preferably, cation exchange column chromatography is selected.

[0014] Preferably, the complexation with copper ions is performed using a copper complexing agent containing copper ions, wherein the copper complexing agent is selected from one or more of copper acetate, copper chloride, and copper dimethylethanol.

[0015] Beneficial effects: Compared with the prior art, the biosynthesis method of blue copper peptide of the present invention, by optimizing the design of fusion protein, obtains fusion protein with better structure and isoelectric point characteristics, which can significantly improve the yield of the final product and has the advantages of low production cost, green and environmentally friendly, and high yield. Attached Figure Description

[0016] Figure 1 : Fusion protein expression results. M: Protein marker; Lane1: Total protein; Lane2: Inclusion body protein; Lane3: Soluble protein;

[0017] Figure 2 HPLC detection results of GHK tripeptide. The retention time of the GHK tripeptide prepared in this invention is consistent with that of the reference standard. Detailed Implementation

[0018] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading this invention, any modifications of the invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Example

[0019] I. Recombinant Protein Design

[0020] The amino acid sequence of the recombinant protein is shown in SEQ ID NO.1. The gene for the protein was obtained by reverse transcription, and its nucleotide sequence is shown in SEQ ID NO.2.

[0021] II. Construction of Recombinant Plasmids

[0022] The gene with the nucleotide sequence shown in SEQ ID NO.2 was synthesized by General Biotechnology (Anhui) Co., Ltd. and inserted between the NcoI and XhoI restriction sites of the pET28a plasmid to construct a recombinant plasmid, which was then sequenced for verification.

[0023] III. Construction of engineered bacteria

[0024] Escherichia coli BL21 (DE3) competent cells were prepared by CaCl2 treatment. The recombinant plasmid was transferred into the cells by heat shock at 42°C. The cells were cultured overnight at 37°C, and single clones were selected for product expression.

[0025] IV. Fermentation Culture

[0026] 1) Shake flask fermentation

[0027] The validated strain was inoculated into 20 ml of LB medium (containing 50 μg / ml kanamycin) and cultured overnight at 37°C as seed culture. This seed culture was then inoculated into 1 L of LB medium (containing 50 μg / ml kanamycin) for fermentation (1% inoculum). Initially, the culture was incubated at 300 rpm and 37°C until OD reached [the desired growth rate]. 600 Once the concentration reaches 0.6, adjust the temperature to 32℃, add IPTG (final concentration 0.4mM) to induce protein expression for 6 hours, and then centrifuge at 8000rpm to collect the bacteria.

[0028] 2) Fermentation in engineered microbial tanks

[0029] The validated strain was inoculated into 100 ml LB medium (containing 50 μg / ml kanamycin) and cultured overnight at 37°C as seed culture. This seed culture was then inoculated into 10 L fermentation medium (containing 50 μg / ml kanamycin, 1% inoculum) and cultured at 37°C. The pH was controlled at 7.0 with concentrated ammonia. Dissolved oxygen was controlled at 30%-40% by adjusting aeration, rotation speed, and glucose supplementation (70% glucose). OD 600 When the temperature reaches 40°C, adjust it to 30°C, add IPTG (final concentration 0.4mM) to start inducing protein expression, and fermentation ends after 10 hours. Centrifuge at 8000 rpm to collect the bacteria.

[0030] The fermentation medium components are as follows:

[0031]

[0032] V. Product Separation and Purification

[0033] Wet bacteria were dissolved in a 1:8 ratio with cell wall disruption buffer (cell wall disruption buffer: 50 mM Tris-Cl, pH 8.0), stirred until no obvious particles were observed, and then sonicated to disrupt the cell wall (sonication conditions: 10-gauge amplitude rod, 700 W, 35 min). After centrifugation, the precipitate was collected to obtain inclusion body protein. The inclusion body protein was washed twice (washing buffer: 50 mM Tris-Cl, pH 8.0, 1% Triton X-100, 10 mM EDTA), and then dissolved in Tris-Cl buffer at pH 9.0.

[0034] VI. Enzyme digestion

[0035] The recombinant protein was cleaved using trypsin. The dissolved inclusion body protein was taken, and trypsin was added at a mass ratio of fusion protein:trypsin = 20:1. The pH was adjusted to 9.0, and the mixture was incubated at 25°C for 6 hours. Subsequently, the precipitate and supernatant were separated by centrifugation at 4°C. The GHK monomer was present in the supernatant, which is the trypsin cleavage product.

[0036] VII. Purification of GHK Tripeptide

[0037] One-step purification was performed using cation exchange chromatography with an SP Sepharose FF column and column material at a 1:1 ratio. The enzyme digestion product was loaded onto a pre-equilibrated SP Sepharose FF column, washed with buffer A until baseline equilibration, and then eluted linearly from 0 to 100% at a volume of 20 CV. The elution peak was collected.

[0038] Buffer A: 50mM Tris-Cl, pH 8.0.

[0039] Buffer B: 50mM Tris-Cl, 500mM NaCl, pH 8.0.

[0040] The elution peak was subjected to nanofiltration desalting to obtain a pure GHK tripeptide solution, which was then lyophilized to obtain the pure product.

[0041] The purity of the prepared GHK monomer samples was further analyzed using HPLC. A Shimadzu-LC-20A dual-pump and SPD-M20A detector were used, and the column was Ultimate. ® An XB-C18 column (5 μm, 4.6 mm x 250 mm) was used, with a detection wavelength of 215 nm. The mobile phase was an aqueous solution of 0.1% acetonitrile (containing 0.1% trifluoroacetic acid). Results showed that the retention time of both the GHK standard and the enzyme-digested GHK monomer was approximately 4.8 min, with a single peak shape and no impurity peaks, proving that the method described in this invention can effectively prepare high-purity GHK monomer. The final GHK monomer yield was calculated to be 3.5 g / L.

[0042] 8. Tripeptide-1 Copper Synthesis

[0043] Dissolve 1.34 g of copper chloride in 30 ml of distilled water, add 3.40 g of GHK tripeptide under stirring, stir at room temperature for 4 h, filter, add 200 ml of ethanol to precipitate solid, filter, wash the filter cake with distilled water and dissolve it in 30 ml of distilled water, add 1.67 ml of hydrochloric acid (6N concentration), stir for 1.5 h, concentrate and freeze dry to obtain blue copper peptide.

[0044] This invention provides a method, and there are many methods and approaches to implement this technical solution. The above description is only a preferred embodiment of this invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technology.

Claims

1. A method for biosynthesis of blue copper peptide, characterized by, The blue copper peptide is obtained by including a recombinant protein according to the amino acid sequence shown in SEQ ID NO. 1, synthesizing a gene encoding the recombinant protein, constructing a recombinant plasmid using the same, then transferring the same into a host bacterium, performing fermentation culture to induce protein expression, separating the protein, performing enzyme digestion and purification, and finally complexing with copper ions.

2. The method of biosynthesis of blue copper peptide according to claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO. 2, or other nucleotide sequences encoding the same protein sequence according to codon degeneracy.

3. The method of biosynthesis of blue copper peptide according to claim 1, characterized in that, The expression plasmid is selected from pET28a, pET30a or pETDuat-1.

4. The method of biosynthesis of blue copper peptide according to claim 1, characterized in that, The host bacterium is selected from E. coli BL21 (DE3) or JM109.

5. The method of biosynthesis of blue copper peptide according to claim 1, characterized in that, The fermentation culture for inducing protein expression uses IPTG as an inducer, the concentration is 0.3-0.5 mM, the induction conditions are 28-40 DEG C, 250-350 rpm for 4-7 h.

6. The method of biosynthesis of blue copper peptide according to claim 1, characterized in that, The method for separating the protein includes breaking the bacterial wall, centrifuging to collect the inclusion body protein, washing the inclusion body protein, and resolubilizing the inclusion body protein.

7. The method of biosynthesis of blue copper peptide according to claim 1, characterized in that, The enzyme digestion is performed at a pH of 8-10, 15-35 DEG C for 4-8 h.

8. The method of biosynthesis of blue copper peptide according to claim 1, characterized in that, The purification method uses ion exchange column chromatography purification method.

9. The method of biosynthesis of blue copper peptide according to claim 1, characterized in that, The complexing with copper ions is performed by using a copper complexing agent containing copper ions, and the copper complexing agent is selected from one or more of copper acetate, copper chloride and copper dimethylate.

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