A soluble expressed mussel myoglobin polypeptide, and preparation method and application thereof

Abstract

The present application relates to a kind of soluble expression of mussel mucin polypeptide and its preparation method and application, the soluble expression of mussel mucin polypeptide is at least one of MAP-PEP1, MAP-PEP2, the amino acid sequence of the MAP-PEP1 as shown in SEQ ID NO.1, the amino acid sequence of the MAP-PEP2 as shown in SEQ ID NO.2.The mussel mucin polypeptide MAP-PEP1, MAP-PEP2 rich in positive charge and DOPA group obtained by the present application can effectively destroy the cell membrane of gram-positive bacteria and gram-negative bacteria, induce oxidative stress response, inhibit bacterial proliferation;Also can promote cell adhesion, migration and proliferation, help wound healing, also have certain efficacy in free radical scavenging, have the great potential to become the next generation of multifunctional bioactive material, provide theoretical basis for being widely used in wound care, implant material, drug delivery, cosmetics and other fields.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a soluble mussel adhesive protein polypeptide and a preparation method and application thereof. BACKGROUND

[0002] Mussel adhesive proteins (MAPs) are a class of biological macromolecules secreted by marine mussels, which have excellent wet-state adhesion properties. The main characteristics are rich in lysine, tyrosine and its derivative 3, 4-dihydroxyphenylalanine (DOPA). These structural units endow the proteins with strong adhesion ability, cross-linking properties and good biocompatibility. Therefore, mussel adhesive proteins show broad application prospects in the field of biomedical materials, such as surgical suturing, tissue engineering, wound dressing, drug delivery systems, cosmetics, etc.

[0003] Among them, the mussel adhesive protein Pvfp-5β (amino acid sequence as shown in SEQ ID NO. 3, and its encoding gene as shown in SEQ ID NO. 6) derived from Perna viridis is a typical mussel adhesive protein, which has attracted widespread attention due to its excellent adhesion properties and biological activity. However, when the full-length protein (such as Pvfp-5β) is expressed in a prokaryotic expression system (such as Escherichia coli), it often exists in the form of insoluble inclusion bodies, which leads to a complex extraction and purification process. The protein with biological activity can be obtained only after denaturation, renaturation and other multi-step treatments (Recombinant mussel protein Pvfp-5: A potential tissue bioadhesive; DOI: 10.1074 / jbc.RA119.009531). This not only increases the complexity and cost of the production process, but also may affect the functional activity of the protein, limiting its further development and clinical application.

[0004] Therefore, it is a technical problem to be solved in the field to develop a mussel adhesive protein polypeptide that can be expressed in a soluble form, retain the key functional domains of mussel adhesive proteins (such as rich in positive charge and DOPA groups), and have good antibacterial and repair activity. SUMMARY

[0005] The purpose of the present application is to provide a soluble mussel adhesive protein polypeptide and a preparation method and application thereof to solve the above problems.

[0006] The present application achieves the above-mentioned purpose by the following technical solutions:

[0007] The application provides a soluble mussel adhesive protein polypeptide, which is derived from mussel adhesive protein Pvfp-5beta and is at least one of MAP-PEP1 and MAP-PEP2, wherein the amino acid sequence of the MAP-PEP1 is shown as SEQ ID NO. 1, and the amino acid sequence of the MAP-PEP2 is shown as SEQ ID NO. 2.

[0008] The application further provides a preparation method of the soluble mussel adhesive protein polypeptide, which comprises the following steps:

[0009] (1) constructing a recombinant protein plasmid of the soluble mussel adhesive protein polypeptide MAP-PEP1 and MAP-PEP2 in vitro, and transforming the recombinant protein plasmid into a protein expression system respectively to express and purify a freeze-dried protein dry powder;

[0010] (2) dissolving the protein dry powder by using a PBS buffer to obtain a protein solution.

[0011] As a further optimization scheme of the application, the protein expression system is E. coli or Pichia pastoris.

[0012] As a further optimization scheme of the application, the concentration of the protein solution is greater than or equal to 3 mg / mL.

[0013] The application further provides an application of the soluble mussel adhesive protein polypeptide in preparation of an antibacterial repair material.

[0014] As a further optimization scheme of the application, the antibacterial bacteria are gram-negative bacteria and gram-positive bacteria.

[0015] As a further optimization scheme of the application, the gram-negative bacteria are E. coli, and the gram-positive bacteria are S. aureus.

[0016] The application has the following beneficial effects:

[0017] 1) The mussel adhesive protein polypeptide MAP-PEP1 and MAP-PEP2 rich in positive charges and DOPA groups prepared by the application can effectively destroy the cell membranes of gram-positive bacteria and gram-negative bacteria, induce oxidative stress response and inhibit bacterial proliferation; the mussel adhesive protein polypeptide can also promote cell adhesion, migration and proliferation, is helpful for wound healing, has certain effects on free radical scavenging, has great potential to become a next-generation multifunctional bioactive material, and provides a theoretical basis for wide application in the fields of wound care, implant materials, drug delivery, cosmetics and the like.

[0018] 2) The soluble expressed mussel adhesive protein polypeptide prepared by the present application can be used for preparing wound dressing material for clinical use, which has the functions of preventing wound infection and promoting wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is the SDS-PAGE map of the soluble expressed mussel adhesive protein MAP-PEP1;

[0020] Figure 2 is the SDS-PAGE map of the soluble expressed mussel adhesive protein MAP-PEP2;

[0021] Figure 3 is the SDS-PAGE map of the mussel adhesive protein Pvfp-5β;

[0022] Figure 4 is the cell morphology map observed under microscope;

[0023] Figure 5 is the cell activity statistical map of the soluble expressed mussel adhesive protein polypeptide MAP-PEP1 and MAP-PEP2;

[0024] Figure 6 is the sample image of the in vitro DPPH free radical scavenging experiment of the soluble expressed mussel adhesive protein polypeptide MAP-PEP1 and MAP-PEP2;

[0025] Figure 7 is the absorbance value of the in vitro DPPH free radical scavenging experiment of the soluble expressed mussel adhesive protein polypeptide MAP-PEP1 and MAP-PEP2;

[0026] Figure 8 is the plate antibacterial effect map of the mussel adhesive protein MAP-PEP1 on E. coli and Staphylococcus aureus;

[0027] Figure 9 is the plate antibacterial effect map of the mussel adhesive protein MAP-PEP2 on E. coli and Staphylococcus aureus. DETAILED DESCRIPTION

[0028] Hereinafter, the present application will be described in further detail with reference to the accompanying drawings. It is necessary to point out here that the following detailed description is only used to further illustrate the present application, and cannot be understood as limiting the scope of protection of the present application, and the skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0029] 1. Materials

[0030] The methods used in this example are conventional methods known to those skilled in the art, and the reagents and other materials used are commercially available products unless otherwise specified.

[0031] 2. Method

[0032] 2.1. Preparation method of mussel adhesive protein polypeptides MAP-PEP1, MAP-PEP2 and Pvfp-5β

[0033] 2.1.1. Preparation of soluble mussel adhesive protein polypeptides MAP-PEP1 and MAP-PEP2

[0034] In the experiments of this application, E. coli expression system was used to prepare mussel adhesive protein polypeptides MAP-PEP1 and MAP-PEP2, but it is not limited thereto, and other protein expression systems such as Pichia pastoris can also be selected for preparation. The method for preparing mussel adhesive protein polypeptides MAP-PEP1 and MAP-PEP2 by E. coli expression system comprises the following steps:

[0035] Step 1: According to the amino acid sequence of mussel adhesive protein polypeptides MAP-PEP1 and MAP-PEP2, the coding genes of mussel adhesive protein polypeptides MAP-PEP1 and MAP-PEP2 were designed and artificially synthesized according to the codon bias of E. coli. The nucleotide sequences of the coding genes are shown in SEQ ID NO. 4-5, respectively. The coding genes of mussel adhesive protein polypeptides MAP-PEP1 and MAP-PEP2 were constructed into recombinant protein expression plasmids in vitro, and the recombinant protein expression plasmids were transformed into E. coli, respectively. The bacterial liquid after induction expression (expression conditions: 0.4 mM IPTG, 20°C induction for 16h) was centrifuged at 4000 rpm, and the bacterial body was collected. The collected bacterial body was resuspended with buffer (the resuspension buffer was PBS buffer with pH 7.4: 20 mM imidazole, 1 mg / mL lysozyme and 1 mM PMSF), and the broken suspension was obtained by high-pressure homogenization using a high-pressure homogenizer;

[0036] Step 2: The broken suspension was purified by Ni-NTA affinity chromatography column. First, low-concentration imidazole (20 mM) buffer was used to wash away impurities, and 0.5 mg / mL tyrosinase was used to modify mussel adhesive protein polypeptides in situ, and then 250 mM imidazole buffer was used to elute the target protein to obtain a crude protein solution. The crude protein solution was dialyzed several times to remove salt and imidazole impurities. The protein solution after dialysis was freeze-dried (freeze-drying conditions: -60°C~-80°C, vacuum degree ~ 15 Pa, 24h) to obtain protein dry powder;

[0037] Step 3: The sample eluted from the nickel column was detected by SDS-PAGE to detect the elution of MAP-PEP1 and MAP-PEP2 proteins, respectively. The experimental results are shown in FIG. 1.Figure 1 、 2 as shown in FIG. 3;

[0038] Step 4: Dissolve the MAP-PEP1 and MAP-PEP2 protein dry powder into PBS buffer solution with pH 7.4 respectively to obtain protein solution with a concentration of 3 mg / mL, and store at -4℃ for standby.

[0039] 2.1.2, Preparation of mussel glycoprotein Pvfp-5β

[0040] In the experiment, the E. coli expression system is used to prepare the mussel glycoprotein Pvfp-5β. In actual operation, other protein expression systems such as Pichia pastoris can also be selected for preparation. The method for preparing the mussel glycoprotein Pvfp-5β by using the E. coli expression system comprises the following steps:

[0041] Step 1: According to the amino acid sequence of the mussel glycoprotein Pvfp-5β, the coding gene of the mussel glycoprotein Pvfp-5β is designed and artificially synthesized based on the codon bias of E. coli. The nucleotide sequence of the coding gene is shown as SEQ ID NO. 6. The coding gene of the mussel glycoprotein Pvfp-5β is constructed into a recombinant protein expression plasmid in vitro, and the recombinant protein expression plasmid is transformed into E. coli. The bacterial liquid after induction expression (the expression conditions are 0.4 mM IPTG, 20℃ induction for 16h) is centrifuged at 4000 rpm to collect the bacterial body. The collected bacterial body is resuspended with a buffer (the resuspension buffer is PBS buffer solution with pH 7.4: 8M urea, 20 mM imidazole, 1 mg / mL lysozyme and 1 mM PMSF), and a high-pressure homogenizer is used for high-pressure homogenization treatment to obtain a broken suspension;

[0042] Step 2: The broken suspension is purified by using a Ni-NTA affinity chromatography column. First, low-concentration imidazole (20 mM) buffer solution is used to wash away impurities. Then, 0.5 mg / mL tyrosinase is used for in-situ modification of the mussel glycoprotein polypeptide. Finally, 250 mM imidazole buffer solution is used to elute the target protein to obtain a crude protein solution. The crude protein solution is dialyzed multiple times to remove salt and imidazole impurities (the dialysis buffer is sequentially PBS pH 7.4 buffer solution (2M urea), PBS pH 7.4 buffer solution, ultrapure water and 5% acetic acid, the dialysis conditions are 4℃, the volume ratio of protein solution to dialysis solution is 1:100, and the dialysis is performed twice). The protein solution after dialysis is freeze-dried (the freeze-drying conditions are -60℃~-80℃, vacuum degree ~15 Pa, and 24h) to obtain protein dry powder;

[0043] Step 3: The sample eluted from the nickel column is detected by SDS-PAGE to detect the protein elution as shown in FIG. 3; Figure 3

[0044] ​Step 4: Dissolve the Pvfp-5β protein dry powder into PBS buffer solution with pH 7.4 to obtain a protein solution with a concentration of 3 mg / mL, and store at -4℃ for standby.

[0045] Experimental conclusion: Figure 1 and Figure 2 The SDS-PAGE of the supernatant and the precipitate of the broken bacterial solution of the full-length protein (mussel adhesive protein Pvfp-5β) is shown in FIG. 4, which shows that there is no protein in the supernatant and a large amount of protein in the precipitate, thus indicating that the full-length protein (mussel adhesive protein Pvfp-5β) is not soluble. Figure 3 The SDS-PAGE of the supernatant and the precipitate of the broken bacterial solution of the full-length protein (mussel adhesive protein Pvfp-5β) is shown in FIG. 4, which shows that there is no protein in the supernatant and a large amount of protein in the precipitate, thus indicating that the full-length protein (mussel adhesive protein Pvfp-5β) is not soluble.

[0046] The sequences of the mussel adhesive protein polypeptides MAP-PEP1 and MAP-PEP2 and the mussel adhesive protein Pvfp-5β are shown in Table 1:

[0047] Table 1 Protein sequence information table

[0048] ;

[0049] 2.2, the recombinant polypeptide has the ability to promote cell migration and diffusion and wound healing

[0050] NIH 3T3 cells were inoculated in a cell culture 6-well plate and cultured overnight in a cell incubator at 37℃ and 5% CO2, then the scratch was observed and recorded at 0h, and then 1% serum, protein (MAP-PEP1, MAP-PEP2, Pvfp-5β; the amount of the three proteins was 0.2 mg / mL), 10% serum was added, respectively, and the scratch healing was observed after 24h and 48h of culture in a cell incubator at 37℃ and 5% CO2, and the experimental data statistics results are shown in Table 2:

[0051] Table 2 Wound healing rate data statistics table of each group

[0052] ;

[0053] The experimental results are as follows: the cell scratch healing rate of the 1% serum treatment group is 12.4%, the cell scratch healing rate of the 10% serum treatment group is 34.3%, which is slightly lower than that of the protein group, compared with the protein Pvfp-5β treatment group, the cell scratch healing rates of the protein MAP-PEP1 treatment group and the protein MAP-PEP2 treatment group are improved, and the improvement rates are 20.2%-36.7%, among which the cell scratch healing rate of the protein MAP-PEP1 treatment group is the most obvious, and the 24h healing rate is 36.5%, the cell scratch healing rate of the protein MAP-PEP2 treatment group is the second, and the 24h healing rate is 32.1%, which indicates that the proteins MAP-PEP1 and MAP-PEP2 have excellent abilities to promote cell proliferation and migration and diffusion, and have the potential to promote wound healing in vitro.

[0054] 2.3, Biocompatibility test of the protein (MAP-PEP1, MAP-PEP2)

[0055] In each well of a 96-well plate, ~10 4 NIH 3T3 cells were inoculated, and after being cultured for 24h, different concentration gradients (0.01-0.5mg / mL) of the protein were added, and the wells without any treatment were used as blank controls, and the cells were cultured for another 24h, and then the cell morphology was observed under a microscope, and the results are shown in Figure 4 Meanwhile, MTT reagent was used to detect the cell activity, and the results are shown in Figure 5 .

[0056] The experimental conclusion is that, as shown in Figure 4 and Figure 5 , the 3T3 cells still maintain good growth state and cell morphology after adding the protein (MAP-PEP1, MAP-PEP2) with a concentration as high as 0.5mg / ml, and it can be found that the protein has a certain promoting effect on the cells, which indicates that the protein has good biocompatibility.

[0057] 2.4, In vitro DPPH free radical scavenging experiment of the protein (MAP-PEP1, MAP-PEP2)

[0058] 500μL of DPPH ethanol solution with a concentration of 0.12mg / mL was mixed with 200μL of the protein (MAP-PEP1, MAP-PEP2), and then an appropriate amount of water or 95% ethanol solution was added to make the total volume of the reaction system reach 2mL. After gently mixing the mixed solution, it was reacted at room temperature for 5 minutes in the dark. Two control groups were set: vitamin E prepared with 95% ethanol solution as the positive control, and pure 95% ethanol solution as the negative control. After the reaction was completed, the samples were first imaged, and the results are shown in Figure 6The absorbance value was then measured at 517 nm wavelength using a SpectraMax Plus 384 microplate reader (Molecular Devices, USA) as shown in the results below. Figure 7 To ensure the reliability of the experimental data, each sample was measured in triplicate.

[0059] Experimental conclusion: from Figure 6 , 7 It can be known that the proteins (MAP-PEP1, MAP-PEP2) have certain scavenging ability for DPPH free radicals, which indicates that the proteins have good antioxidant activity.

[0060] 2.5, in vitro antibacterial test of protein (MAP-PEP1, MAP-PEP2)

[0061] The frozen strains of E. coli and S. aureus were inoculated into 5 mL of fresh culture medium, and cultured at 37°C, 220 rpm on a shaker for 16 h. The concentration of the cultured bacteria was adjusted to OD 600 0.1, 10 μL of protein (MAP-PEP1, MAP-PEP2) was added to 100 μL of the above bacterial solution, and incubated at 37°C for 1 h. The blank control was not added with protein. The incubated bacterial solution was diluted 10000 times, 100 μL of the diluted bacterial solution was plated, and the colony morphology was observed after 24 h of culture at 37°C. The colony count was performed, and the photographs were recorded. The results are shown in Figure 8 , Figure 9 .

[0062] Experimental conclusion: from Figure 8 , Figure 9 It can be known that the proteins (MAP-PEP1, MAP-PEP2) have significant inhibitory effect on gram-negative and gram-positive bacteria.

[0063] In summary, the soluble mussel myoglobin polypeptides MAP-PEP1 and MAP-PEP2 rich in positive charges and DOPA groups can effectively destroy the cell membranes of gram-positive bacteria and gram-negative bacteria, induce oxidative stress, and inhibit bacterial proliferation; can promote cell adhesion, migration and proliferation, and help wound healing, and also have certain effect in free radical scavenging.

[0064] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the protection scope of the present application.

Claims

1. A soluble, expressed Mytilus mussels mucoprotein polypeptide, characterized in that, The soluble expressed mussel myoglobin polypeptide is at least one of MAP-PEP1 and MAP-PEP2, the amino acid sequence of the MAP-PEP1 is shown as SEQ ID NO. 1, and the amino acid sequence of the MAP-PEP2 is shown as SEQ ID NO.

2.

2. A method of producing a soluble expressed Mytilus mussels mucus polypeptide according to claim 1, wherein, The method comprises the following steps: (1) constructing a recombinant protein plasmid of the soluble expressed mussel myoglobin polypeptide MAP-PEP1 and MAP-PEP2 in vitro, and transforming the recombinant protein plasmid into a protein expression system respectively to express and purify a freeze-dried protein dry powder; (2) dissolving the protein dry powder by using a PBS buffer to obtain a protein solution.

3. A method of producing a soluble expressed Mytilus musselsin mucus polypeptide according to claim 2, wherein, The protein expression system is Escherichia coli or Pichia pastoris.

4. The method of claim 2, wherein the soluble expressed Mytilus moolypeptide is prepared by the steps of: The concentration of the protein solution is greater than or equal to 3 mg / mL.

5. Use of a soluble expressed Mytilus mussels' mucoprotein polypeptide according to claim 1 for the preparation of an antibacterial, reparative material, characterized in that, The antibacterial fungi are gram-negative bacteria and gram-positive bacteria; the repair is to promote wound healing and eliminate DPPH free radicals.

6. Use according to claim 5, characterized in that, The gram-negative bacteria are Escherichia coli, and the gram-positive bacteria are Staphylococcus aureus.

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