Novel method for realizing efficient biosynthesis of copper-blue peptide

Abstract

The invention relates to a biosynthesis method of cosmetic tripeptide GHK (glycine-histidine-lysine), which is characterized in that a GHK oligopeptide is prepared by carrying out microbial fermentation synthesis and trypsin specific hydrolysis on artificially designed GHK multiple artificial sequences. And on the basis of the original sequence, the GHK yield is improved by adding a specific interval sequence. Compared with a traditional chemical synthesis method, the GHK oligopeptide biosynthesis method has the advantages that raw materials are cheap and simple, steps are easy to implement, and the GHK oligopeptide biosynthesis method is environmentally friendly and beneficial to improving the application level of GHK tripeptide in the field of beauty makeup.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and in particular relates to a biosynthetic method of a GHK (glycine-histidine-lysine) tripeptide. Background Art

[0002] Blue copper peptide (GHK-Cu) is a tripeptide-copper complex. The tripeptide GHK composed of glycine, histidine and lysine has a high affinity with divalent copper ions, and the two can spontaneously form a complex blue copper peptide when they meet. To date, blue copper peptide has been confirmed by many studies to have multiple functions, including helping DNA repair, anti-inflammation, anti-aging, and wound repair. As a mild ingredient, blue copper peptide has been used in skin care products for many years and has high application value. At present, there are many preparation methods, mainly including chemical synthesis and microbial synthesis. Chemical synthesis is the mainstream method, but it has the disadvantages of more by-products, low yield, difficult purification, high cost, and environmentally unfriendly materials in the production process. Based on genetic engineering methods, the biosynthesis of blue copper peptides through repetitive sequence expression has been a research hotspot in recent years, but there are currently problems such as low expression levels, which are difficult to meet the needs of industrial production. Therefore, for the biosynthesis method, exploring new methods to increase the expression of GHK to reduce the production cost of blue copper peptides is of great significance for promoting the application of blue copper peptides. It is expected that this invention will expand the application market scale of blue copper peptides, making this excellent anti-aging ingredient available to more people. Summary of the invention

[0003] The purpose of the present invention is to synthesize a short peptide GHK using a biological method. The yield of GHK is increased by designing a tandem repeat sequence. We found that GHK, as a hydrophilic short peptide, has a very low yield when expressed in tandem directly. By adding a specific spacer sequence YSDEPR on the basis of the repeat sequence, the expression amount of the GHK tandem polypeptide can be effectively increased.

[0004] The biosynthesis method of GHK is as follows:

[0005] (1) An artificial amino acid sequence containing a GHK repeating sequence was designed and named the GHK multiple sequence. Its characteristic is the presence of multiple repeated GHK sequences. When the number of GHK repeats is relatively large, in order to reduce the overall hydrophilicity of the sequence, a hydrophobic amino acid sequence such as YSDEPR is inserted between some AAPF repeating sequences, thereby facilitating the normal expression of the artificial sequence in microorganisms.

[0006] (2) entrusting a gene synthesis company to synthesize the designed GHK multiple sequence DNA sequence and integrate it into a microbial expression plasmid, preferably the Escherichia coli pET series as the expression plasmid;

[0007] (3) Introducing the expression plasmid into an engineering strain to ferment and express the polypeptide molecules of the GHK multiple sequences, preferably selecting Escherichia coli as the engineering strain.

[0008] (4) adding trypsin to the prepared GHK multiple sequence polypeptides for incubation and hydrolysis, obtaining GHK short peptides by ultrafiltration, and confirming the synthesis of the GHK product by liquid chromatography-mass spectrometry analysis;

[0009] The beneficial effects of the present invention are:

[0010] The invented GHK uses a biosynthetic method. Compared with the chemical method, it does not require high-purity amino acids and catalysts as synthetic raw materials. The steps are simple and easy to implement, and the production process is green and environmentally friendly. It is expected to significantly reduce production costs in the future, thereby promoting the application of GHK in the cosmetics industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 , Liquid chromatography-mass spectrometry analysis of the product of trypsin hydrolysis after recombinant expression of GHK-15 repeat sequence polypeptide containing YSDEPR sequence interval. The upper figure is the chromatogram, and the lower figure is the mass spectrum of GHK. DETAILED DESCRIPTION

[0012] The technical content of the present invention is further described below in conjunction with the embodiments, but the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited by the following embodiments.

[0013] Example 1: Design of GHK multiple sequences

[0014] Two GHK multiple protein sequences were designed, protein sequence 1 contained 15 GHK sequences and was named 15-GHK. Protein sequence 2 inserted a hydrophobic sequence within a certain interval within the 15 GHK sequences and was named 15in-GHK.

[0015] 15-GHK protein sequence:

[0016] GHKGHKGHKGHKGHKGHKGHKGHKGHKGHKGHKGHKGHKGHKGHKGHK

[0017] 15in-GHK protein sequence:

[0018] GHKGHKGHKYSDEPRGHKGHKGHKYSDEPRGHKGHKGHKYSDEPRGHKGHKGHKYS

[0019] DEPRGHKGHKGHK

[0020] Example 2: Biosynthesis of GHK

[0021] 1. Entrust a gene synthesis company to synthesize the DNA sequences of 15-GHK and 15in-GHK and insert them into the pET28a(+) plasmid.

[0022] 2. Transform the constructed plasmid into E. coli BL21 (DE3)

[0023] (1) Take out competent cells (80 μL) from -80°C and thaw in an ice bath.

[0024] (2) Add 10 μL of the ligation product or 2-5 μL of the plasmid to be transformed to the competent cells, mix gently, and place on ice for 30 min.

[0025] (3) Heat shock at 42°C in a water bath for 30 seconds and place in an ice bath for 2 minutes.

[0026] (4) Add 900 μL of LB medium preheated at 37°C and culture at 37°C in a shaker at 220 rpm for 1 hour.

[0027] (5) Collect the cells by centrifugation at 4000 r / min for 5 min, discard part of the supernatant and resuspend the cells, take an appropriate amount and spread on an Amp (ampicillin)-resistant LB plate, place the plate in a 37°C incubator and culture overnight for 12-16 h.

[0028] 3. Fermentation induction culture of the transformed strain after the plasmid is introduced

[0029] (1) The bacterial solution was transferred to LB liquid medium containing Amp (ampicillin) resistance (final Amp concentration was 100 μg / mL) and cultured at 37°C and 220 r / min until the bacterial solution OD600 = 0.6-0.8.

[0030] (2) Add IPTG (final concentration 0.5 mmol / L) and induce culture at 16°C and 120 r / min for 16-20 h.

[0031] 4. Treatment of fermentation broth

[0032] The fermented bacterial liquid was centrifuged at 12000r / min for 30min and the precipitate was collected. The precipitate was crushed by a tissue grinder. The crushing parameters of the tissue grinder were: crushing time 45s; interval time 15s; power 60w; total time 30min; temperature 4°C.

[0033] 5. Incubate and hydrolyze to obtain GHK short peptide.

[0034] An appropriate amount of trypsin was added to the broken bacterial solution and hydrolyzed at 37°C for 3 h, and the GHK short peptide was obtained after ultrafiltration.

[0035] 7. Detect the hydrolyzed peptides by mass spectrometry.

[0036] The hydrolyzate was detected by Agilent liquid chromatography time-of-flight mass spectrometry, the column model was XBridgepeptide BEH C18 (2.1 mm x 150 mm, 3.5 μm), the mobile phase gradient elution conditions were shown in Table 1, and the mass spectrometer detector parameters included energy: 4500 v, slope: 3.6, offset: 4.8, Gas Temp (°C): 325, Gas flow (L / min): 13, flow rate: 0.20 ml / min. The mobile phase was a 5% acetonitrile solution with 0.1% formic acid added.

[0037] The test results showed that the GHK fermentation preparation level of the control without adding the spacer sequence was only 0.1 mg / mL, while after adding the spacer sequence, the GHK fermentation preparation level increased to 0.3 mg / mL, which is 3 times that of the control. The results of liquid chromatography-mass spectrometry test proved that adding the spacer sequence in the tandem sequence increased the production of AAPF short peptides. The results are shown in the attached figure. Figure 1 shown.

Claims

1. A new method for achieving efficient biosynthesis of blue copper peptides through repetitive sequence expression, the core feature of which is: inserting an appropriate proportion of repetitive spacer sequences YSDEPR into the artificially designed GHK multiple expression sequence, thereby achieving efficient expression and preparation of GHK in the engineered strain.

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