A biosynthetic method for recombinant transdermal cyclic peptide and its transdermal absorption application

Abstract

The present invention belongs to the field of recombinant protein engineering, and specifically relates to a biosynthesis method of a recombinant transdermal cyclic peptide and a transdermal absorption application. The biosynthesis method of the recombinant transdermal cyclic peptide of the present invention comprises the following steps: (1) constructing a tandem repeat sequence of a TDP gene fragment into an Escherichia coli expression vector, and transferring it into Escherichia coli for expression, so as to obtain a recombinant transdermal cyclic peptide; (2) releasing the TDP fragment by pepsin cleavage. The present invention provides an effective method for synthesizing cTDP in an engineered bacterial recombinant expression system. The method cleverly introduces a pepsin site, has a high enzymatic cleavage efficiency, can use low-cost pepsin to achieve polypeptide enzymatic cleavage, and does not produce redundant amino acids. The obtained polypeptide is consistent with the designed molecular structure, and can be used to obtain a variety of active polypeptides in large quantities and at low cost.

Description

Technical Field

[0001] The invention belongs to the field of recombinant protein engineering, and specifically relates to a biosynthesis method of a recombinant transdermal cyclic peptide and its transdermal absorption application. Background Art

[0002] As a cyclic polypeptide that can enhance the transdermal absorption of drugs, transdermal cyclic peptides have shown great application prospects in the field of drug delivery, especially in the transdermal delivery of macromolecular drugs. Under the current technical background, small molecule polypeptides are mainly synthesized by chemical methods. However, when using chemical synthesis methods to synthesize long peptides with a length of more than 10 amino acid residues, or to synthesize complex polypeptides containing multiple disulfide bond structures, many problems are faced. On the one hand, the cost is extremely expensive and the cost of chemical synthesis is high, which greatly limits its wide application. On the other hand, a large amount of organic solvents will be used in the chemical synthesis process, which not only puts great pressure on the environment and is not green and environmentally friendly, but may also bring residual risks in the subsequent processing process, affecting the quality and safety of the final product.

[0003] With the continuous advancement of science and technology, people have higher and higher requirements for drug delivery systems. They hope to achieve efficient transdermal absorption of drugs, and also hope that the synthesis method is green, environmentally friendly and cost-controlled. Driven by such demands, it is imperative to find a new, more efficient and environmentally friendly method for synthesizing transdermal cyclic peptides. It is in this context that the present invention adopts a synthetic biology method, uses DNA recombination technology to optimize the transdermal peptide sequence, and cleverly uses a single amino acid as a protease cleavage site to splice the repeated sequence in series, thereby forming a recombinant transdermal peptide (rTDP) that can be obtained by microbial synthesis in a long sequence. Through a specific protease cleavage reaction, the repeated polypeptide is sheared and released. This process does not produce an excessively long cleavage site or no redundant amino acids at all, has little effect on the polypeptide structure, and is convenient for subsequent further extraction. At the same time, the process of the invention does not use any toxic and harmful organic solvents, is very green and environmentally friendly, and opens up a new path for the synthesis and application of transdermal cyclic peptides. Summary of the invention

[0004] The present invention moves the alanine A at the first position of the transdermal peptide ACSSSPSKHCG to the end, and adds phenylalanine F at the end. As a result, it is found that the transdermal peptide tandem sequence can be cleaved by pepsin, and phenylalanine will be cleaved, so that the sequence does not produce redundant amino acids. Therefore, the present invention constructs a tandem repeat sequence of the TDP gene fragment in this way and incorporates it into the Escherichia coli expression vector pET-9a (+). The recombinant plasmid is transformed into Escherichia coli, and the (TDP)n fusion protein is expressed in high yield. It is purified by Ni affinity chromatography, and then cleaved by pepsin to release the TDP fragment, and finally cyclized by oxidation to form cTDP. A fusion protein with a yield of at least 500 mg / L is obtained in the fermentation of the recombinant strain. The transdermal absorption of green fluorescent protein can be significantly promoted by mixing with green fluorescent protein and applying it topically on rat skin. In general, the invention provides an effective method for synthesizing cTDP in an engineered bacterial recombinant expression system. The method cleverly introduces a pepsin site, has a high enzymatic cleavage efficiency, can use cheap pepsin to achieve polypeptide enzymatic cleavage, and does not produce excess amino acids. The obtained polypeptide is consistent with the designed molecular structure, and can be used to obtain a variety of active polypeptides in large quantities and at low cost. Therefore, the first object of the present invention is to provide a biosynthetic method for a recombinant transdermal cyclic peptide. The second object of the present invention is to provide a recombinant transdermal cyclic peptide. The third object of the present invention is to provide an application of a recombinant transdermal cyclic peptide.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] As a first aspect of the present invention, a biosynthetic method of a recombinant transdermal cyclic peptide comprises the following steps:

[0007] (1) constructing a tandem repeat sequence of a TDP gene fragment into an E. coli expression vector, and transferring the vector into E. coli for expression to obtain a recombinant transdermal cyclic peptide; wherein:

[0008] The steps of constructing the tandem repeat sequence of the TDP gene fragment include: adding aromatic amino acids or leucine to the end of the transdermal peptide, and the front end of the transdermal peptide does not contain basic amino acids;

[0009] The alanine A at the first position of the transdermal peptide ACSSSPSKHCG is moved to the end, and a phenylalanine F is added to the end; and / or,

[0010] The alanine A at the first position of the transdermal peptide ACSSKKSKHCG is moved to the end, and a phenylalanine F is added to the end; and / or,

[0011] Add phenylalanine F to the end of the transdermal peptide CTGSTQHQCG, and / or

[0012] Phenylalanine F was added to the end of the transdermal peptide CKTGSHNQCGF.

[0013] (2) Pepsin cleavage releases TDP fragments.

[0014] Furthermore, the aromatic amino acid is one of phenylalanine F, tryptophan W and tyrosine Y, etc.

[0015] Preferably, the tandem repeat sequence is greater than or equal to 2.

[0016] Preferably, the method further comprises a Ni affinity chromatography purification step.

[0017] Preferably, the method further comprises the step of cyclization to form cTDP by oxidation.

[0018] The amino acid sequence of the recombinant transdermal peptide is: F (CSSSPSKHCGAF) n 、F(CSSKKSKHCGAF) n 、F(CTGSTQHQCGF) n or F(CKTGSHNQCGF) n , where n≥2.

[0019] The present invention also provides an application of the above-mentioned biosynthesis method, wherein the application is one or more of a) to c);

[0020] a) preparing recombinant transdermal cyclic peptides;

[0021] b) preparing a drug for transdermal administration;

[0022] c) Preparation of cosmetics.

[0023] The present invention also provides a recombinant transdermal cyclic peptide, which is prepared by the above method.

[0024] Finally, the present invention provides an application of the above-mentioned recombinant transdermal cyclic peptide, wherein the application is for preparing a drug for transdermal administration.

[0025] Compared with the prior art, the present invention has at least the following beneficial effects:

[0026] In the present invention, the characteristics of several transdermal cyclic peptide sequences are combined, and pepsin, which can be used for phenylalanine or leucine, is added between the sequences for enzymatic cleavage. At present, since the amino acid types in several transdermal peptides are simple, there are no aromatic amino acids and leucine, but some basic amino acids are contained. By utilizing this characteristic for sequence design, accurate cleavage of polypeptide fusion fragments can be achieved, and no redundant amino acid residues can be achieved;

[0027] The present invention constructs a tandem repeat sequence of a TDP gene fragment and incorporates it into an Escherichia coli expression vector pET-9a (+). The recombinant plasmid is transformed into Escherichia coli, and the (TDP)n fusion protein is expressed in high yield. The TDP fragment is purified by Ni affinity chromatography, then cleaved by pepsin to release it, and finally cyclized by oxidation to form cTDP. A yield of at least 500 mg / L of fusion protein is obtained in the fermentation of the recombinant strain. The transdermal absorption of green fluorescent protein can be significantly promoted by mixing it with green fluorescent protein and applying it topically to the skin of rats. In general, the invention provides an effective method for synthesizing cTDP in an engineered bacterial recombinant expression system, which does not produce redundant amino acids, is consistent with the designed molecular structure, and can be used to obtain a variety of active polypeptides in large quantities and at low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the fermentation expression level of recombinant transdermal peptide;

[0029] Figure 2 is the HPLC spectrum of cTDP-1;

[0030] Figure 3 is the HPLC spectrum of cTDP-2;

[0031] Figure 4 is the HPLC spectrum of cTDP-3;

[0032] Figure 5 is the HPLC spectrum of cTDP-4;

[0033] Figure 6 This is a diagram of the experiment on how transdermal cyclic peptide promotes green fluorescent protein penetration;

[0034] Figure 7 Figure 1 is a cytotoxicity assay diagram;

[0035] Figure 8 This is a 30-minute stability test diagram;

[0036] Fig. 9 This is a graph of a 6-hour stability experiment. DETAILED DESCRIPTION

[0037] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0038] Example 1 Transdermal Cyclic Peptide Sequence Optimization

[0039] According to the enzymatic properties of each protease, three commonly used proteases were designed, namely pepsin, trypsin and TEV protease. In this example, we designed n=3 enzymatic fragments, and analyzed the enzymatic products according to their sequence composition and the properties of the protease. The results are shown in the following table. The sequence of the transdermal cyclic peptide TD1 before optimization is: ACSSSPSKHCG.

[0040] Table 1 Transdermal peptide tandem sequence digestion products

[0041] Concatenation sequence Pepsin cleavage products Trypsin cleavage products TEV protease cleavage products (ACSSSPSKHCG)3 N / A ACSSSPSK, HCGACSSSPSK, HCGACSSSPSK, HCG N / A (ACSSSPSKHCGF)3 ACSSSPSKHCGF, ACSSSPSKHCGF, ACSSSPSKHCGF ACSSSPSK, HCGFACSSSPSK, HCGFACSSSPSK, HCGF N / A (ACSSSPSKHCF)3 N / A ACSSSPSK, HCFACSSSPSK, HCFACSSSPSK, HCF N / A (CSSSPSKHCGAF)3 CSSSPSKHCGA, F, CSSSPSKHCGA, F, CSSSPSKHCGA, F CSSSPSK, HCGAFCSSSPSK, HCGAFCSSSPSK, HCGAF N / A N / A:not available

[0042] The results showed that TEV protease could not cleave without the presence of a specific cleavage site, and trypsin would cleave from the K-terminus in the middle of the sequence, thereby destroying the polypeptide sequence (fragments are separated by ","). Pepsin could not cleave without adding amino acids, but it could cleave at the phenylalanine terminus after adding phenylalanine at the end, but phenylalanine would be introduced. If an amino acid in the sequence is removed, such as glycine G at position 11, it cannot be cleaved by pepsin. If alanine A at position 1 is moved to the end and phenylalanine F is added to the end, it can be cleaved by pepsin, and phenylalanine will be cleaved, so that no extra amino acids are produced in the sequence.

[0043] Example 2 Expression and purification of recombinant transdermal cyclic peptide

[0044] Pepsin was used according to the method of Example 1. Four polypeptide sequences, cTDP_1, cTDP_2, cTDP_3 and cTDP_4, were designed in this example, and their biological activities will be further studied in the following examples.

[0045] According to the analysis of the enzyme cleavage products in Example 1, in the present invention, several groups of tandem sequences of transdermal peptide sequences were designed, and the tandem method is shown in Table 2:

[0046] Table 2 Tandem sequence of transdermal peptide sequences

[0047] Concatenation sequence Sequence after restriction digestion Number of amino acids <![CDATA[SEQ ID NO.1:F(CSSSPSKHCGAF) n (n≥2)]]> cTDP_1:CSSSPSKHCGA 11 <![CDATA[SEQ ID NO.2:F(CSSKKSKHCGAF) n (n≥2)]]> cTDP_2:CSSKKSKHCGA 11 <![CDATA[SEQ ID NO.3:F(CTGSTQHQCGF) n (n≥2)]]> cTDP_3:CTGSTQHQCG 10 <![CDATA[SEQ ID NO.4:F(CKTGSHNQCGF) n (n≥2)]]> cTDP_4:CKTGSHNQCG 10 SP:ACNATLPHQCG SP:control peptide

[0048] The nucleic acid sequence of the recombinant transdermal cyclic peptide rTDP (SEQ ID NO.1-4) was designed. In this embodiment, the tandem sequence n=5, 10, 25 was selected, and 6 histidine tag sequences and stop codons were added after each sequence. The synthesis of the sequence was performed by General Biotechnology (Anhui) Co., Ltd. The synthesized nucleic acid fragment was inserted into the pET9a vector through the Nde I and BamH I sites and transferred into the protease-deficient host strain BL21 (DE3) plysS competent cells. The recombinant clones were screened by LB medium containing kanamycin. Cultured overnight at 37°C. The monoclonal strains were screened the next day and transferred to fresh LB medium for culture at 37°C and 220rpm to the logarithmic growth phase. IPTG was added at a ratio of 1:1000 and cultured at 37°C for 3 hours. The bacteria were collected at 5000rpm. The expression of the fusion polypeptide was semi-quantitatively determined by SDS-PAGE electrophoresis, see Table 3.

[0049] Table 3 Expression of TDP (mg / L)

[0050] TDP_1 TDP_2 TDP_3 TDP_4 n=5 50 80 80 80 n=10 200 200 200 200 n=20 450 450 480 450 n=25 500 600 550 500

[0051] Figure 1 It shows that (TDP_1)n, (TDP_2)n, (TDP_3)n and (TDP_4)n are all expressed in Escherichia coli. As n increases, the expression level increases. After n>20, the growth rate of the expression level decreases significantly. In addition, since the difficulty of DNA synthesis of fusion polypeptides with n>25 increases significantly, the embodiment adopted by the present invention takes n=25 as the research object. When n is 25, the molecular weights of the four fusion proteins are 32.58 kDa, 30.78 kDa, 30.06 kDa, and 29.74 kDa, respectively. However, it is not difficult to find from the phenomenon that as n increases, its expression level will also increase to a certain extent. The average expression concentration of the fusion protein is between 0.5-1.5 g / L. The bacteria expressing rTDP were washed with PBS (pH = 7.4, NaCl: 8 g / L, Na 2 HPO 4 :2.86 g / L,KH 2 PO 4:0.2 g / L, KCl:0.19g / L) resuspended, 800bar high pressure crushed, 10000rpm high speed centrifugation to separate supernatant and precipitate. The fusion protein forms inclusion bodies, so the inclusion bodies are collected, washed three times with PBS, dissolved with 8M urea, purified by Ni metal chelate chromatography under denaturing conditions, and 1mM DTT was added after purification to completely open the disulfide bonds in the solution. SephroseG25 desalting column (purchased from cytivia) was then used to remove imidazole and urea, and the buffer solution was replaced with 20mM sodium acetate, pH2.2. Aktapurifer was used for detection and collection in this process, and 215nm wavelength was used for detection, and the front peak was collected.

[0052] The following examples are all carried out using n=25.

[0053] Example 3 Transdermal Cyclic Peptide Enzymatic Cleavage and Cyclization

[0054] Add 0.01% pepsin and digest overnight at 4°C. After digestion, use sephrose G25 desalting column to remove large molecular proteins, collect tail peaks, slowly introduce air into the peptide solution, and stir overnight at 4°C to cyclize to form cTDP. Freeze-dry the cTDP solution. Dissolve cTDP in water to a certain concentration, separate using C-18 reverse phase chromatography column, and obtain Figure 2-Figure 5 The HPLC test spectrum of the 1.0 mg / mL sample shown is as follows:

[0055] The mobile phases were A: water + 0.1% TFA, B: acetonitrile + 0.1% TFA;

[0056] The main peak was detected and collected at a volume ratio of mobile phase A: mobile phase B = 70:20, a flow rate of 1 mL / min, an injection volume of 20 μL, and a detection wavelength of 220 nm.

[0057] Example 4 Transdermal Cyclic Peptide Penetration Enhancement Experiment

[0058] The ex vivo skin of the rat abdomen was removed by hair removal, and the conductivity of the skin was measured to ensure the integrity of the skin barrier. Skin samples with a resistivity higher than 50 kΩ were used in the experiment. The skin was clamped in a Franz diffusion cell. The receptor chamber below the diffusion cell was filled with PBS solution, and 100 μg of cTDP and 100 μg of green fluorescent protein GFP (expressed and purified by our company) were mixed in 1 mL of PBS solution. 200 μL of the mixed solution was placed in the donor chamber on the upper layer of the diffusion cell. 100 μL of the sample to be tested was collected from the receptor chamber at 30 minutes, 2 hours, 4 hours, 8 hours and 12 hours. The protein content was quantitatively detected using a GFP ELISA kit (purchased from Boster Biotechnology, Cat. No.: EK1440). For results, see Figure 6, Transdermal cyclic peptides promote green fluorescent protein penetration.

[0059] Example 5 Cytotoxicity Assay

[0060] The effect of synthetic peptides on cell viability was measured at selected concentrations using the methylthiazolyldiphenyltetrazolium bromide (MTT) assay. 3T3 cells (2 × 10 4 Cells were plated in 96-well tissue culture plates at 400 μg / mL and 200 μmol / mL, respectively, for 24 hours. cTDP was dissolved in DMEM cell culture medium at concentrations of 0.5 μmol / mL and 5.0 μmol / mL, respectively. The cells were cultured with 200 μL of the four cTDP solutions or DMEM for 24 h as blank controls. Thereafter, 500 μg / mL MTT was added, and the plate was incubated for another 4 hours. The culture medium was then removed and replaced with 200 μL of dimethyl sulfoxide (DMSO), and the optical density (OD) was measured at 550 nm using a microplate reader (SpectraMax Plus384, Molecular Devices, USA). The percentage of cell viability was calculated based on the absorbance measured relative to the absorbance of cells exposed to the control group. The results are shown in Figure 7 It was shown that compared with the blank group, the cell survival rate remained above 95% in the presence of the four cTDPs, and there was no significant toxicity to the cells.

[0061] Example 6 Stability Analysis of cTDP

[0062] This example studies whether cTDP is unstable or degraded by enzymes in the tissue when tested in vitro and in skin tissue. In the in vitro experiment, the cyclized cTDP was dissolved in PBS solution and kept in a 37°C water bath for 30 minutes and 6 hours. In the in vivo experiment, fresh rat skin was cut into 2×2 cm 2 , mix the skin PBS dilution with a homogenizer and homogenize it, take 1 ml of the skin homogenate, add cTDP to a final concentration of 500 ppm, stir continuously (200 rpm) and keep in a controlled water bath (37°C). After 30 minutes and 6 hours, take samples from the solution and dilute them with water containing 0.1% (v / v) trifluoroacetic acid to stop degradation. All tests were performed in triplicate. After high-speed centrifugation, the supernatant was taken and the cTDP content was quantified by HPLC. Figure 8 The results showed that after incubation with skin homogenate, the contents of the four cTDPs and SP did not decrease significantly within 30 minutes, while the linearized TDP was significantly degraded. The degradation rate of linearized TDP_4 reached 40%. In the in vitro experiment, cTDP was almost not degraded. Compared with the linearized transdermal peptide, the cyclized cTDP had higher stability. However, after 6 hours ( Fig. 9), the four cTDP contents and SP were significantly degraded, indicating that cTDP will be slowly decomposed by enzymes when it enters the skin tissue and will not remain or accumulate in the body. cTDP did not degrade in the in vitro experiment, indicating that it has good stability. As a polypeptide carrier that promotes the transdermal absorption of macromolecules, cTDP can be degraded by the human body to avoid the accumulation of exogenous substances in the body, and has better application safety.

[0063] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A biosynthetic method for a recombinant transdermal cyclic peptide, characterized in that: The steps include: (1) constructing the tandem repeat sequence of the TDP gene fragment into an E. coli expression vector and transferring it into E. coli for expression to obtain a recombinant transdermal cyclic peptide; The tandem repeat sequence of the TDP gene fragment is: F (CSSSPSKHCGAF) n 、F(CSSKKSKHCGAF) n 、F(CTGSTQHQCGF) n or F(CKTGSHNQCGF) n , where n≥2; The steps of constructing the tandem repeat sequence of the TDP gene fragment include: adding aromatic amino acids or leucine to the end of the transdermal peptide, and the front end of the transdermal peptide does not contain basic amino acids; The alanine A at the first position of the transdermal peptide ACSSSPSKHCG is moved to the end, and a phenylalanine F is added to the end; and / or, The alanine A at the first position of the transdermal peptide ACSSKKSKHCG is moved to the end, and a phenylalanine F is added to the end; and / or, Adding phenylalanine F to the end of the transdermal peptide CTGSTQHQCG; and / or, Add phenylalanine F to the end of the transdermal peptide CKTGSHNQCG; (2) Purification by Ni affinity chromatography followed by pepsin cleavage to release the TDP fragment; (3) Cyclization to form cTDP by oxidation.

2. The biosynthesis method according to claim 1, characterized in that The amino acid sequence of the constructed recombinant transdermal cyclic peptide is: CSSSPSKHCGA, CSSKKSKHCGA, CTGSTQHQCG or CKTGSHNQCG.

3. An application of the biosynthesis method according to claim 1 or 2, characterized in that: The application is one or more of a) to c); a) preparing recombinant transdermal cyclic peptides; b) preparing medicaments for transdermal administration; c) Preparation of cosmetics.

4. A recombinant transdermal cyclic peptide, characterized in that: The recombinant transdermal cyclic peptide is prepared by the method according to claim 1.

5. A use of the recombinant transdermal cyclic peptide as claimed in claim 4, characterized in that: The application is to prepare drugs or cosmetics for transdermal administration.

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