Recombinant silk fibroin peptide as well as preparation method and application thereof

Abstract

The invention discloses recombinant silk fibroin peptide as well as a preparation method and application thereof, and belongs to the technical field of cosmetic raw material development. The amino acid sequence of the recombinant silk fibroin peptide SFHR6 disclosed by the invention is as shown in SEQ ID NO. 1. Soluble expression of the recombinant silk fibroin peptide is achieved through prokaryotic expression and eukaryotic expression, the high-purity soluble recombinant silk fibroin peptide SFHR6 is obtained through nickel column affinity chromatography, hydrophobic chromatography, cation chromatography and anion chromatography technologies, and the recombinant silk fibroin peptide SFHR6 is high in purity, high in tyrosinase inhibition rate and strong in whitening effect. The purification process provided by the invention is simple and convenient, has good repeatability, effectively solves the quality control problem of naturally extracted protein products, has the advantages of high efficiency, high-purity heterologous expression and low large-scale production cost, and has relatively high industrial application potential.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetic raw material development, and in particular to a recombinant silk fibroin peptide and a preparation method and application thereof. Background Art

[0002] As a natural polymer material, silk fibroin is widely used in biomedicine, tissue engineering, drug delivery and new functional materials due to its excellent mechanical properties, biocompatibility and degradability. However, silk fibroin is currently mainly extracted from natural silk. This traditional method of obtaining silk fibroin is not only limited by natural resources, but also has a complex extraction process and low efficiency, making it difficult to meet the needs of industrial production. In addition, natural silk fibroin is difficult to control in terms of purity and consistency of performance, further limiting its potential in high-end applications. Therefore, the synthesis of silk fibroin in heterologous hosts through recombinant technology provides a new solution for large-scale production.

[0003] Although the production technology of recombinant silk fibroin has gradually matured, the existing methods generally have the problems of cumbersome process, high cost and high technical requirements in the expression and purification process, and the prepared recombinant silk fibroin also generally has the problems of low purity and weak efficacy. These problems make the actual operation of large-scale production of recombinant silk fibroin more difficult, and the promotion and application are limited. Therefore, it is necessary to provide a preparation method of recombinant silk fibroin that is simple to operate, high in purity and suitable for large-scale production. Summary of the invention

[0004] The object of the present invention is to provide a recombinant silk fibroin peptide and a preparation method and application thereof, to solve the problems existing in the above-mentioned prior art. The present invention realizes the soluble expression of the recombinant silk fibroin peptide by prokaryotic expression and eukaryotic expression, and obtains a highly purified soluble recombinant silk fibroin peptide SFH_R6 by nickel column affinity chromatography, hydrophobic chromatography, cation chromatography and anion chromatography technology, and the recombinant silk fibroin peptide SFH_R6 has high purity, high tyrosinase inhibition rate, and strong whitening effect. The purification process of the present invention is simple and reproducible, and the quality control problem of natural extracted protein products is effectively solved, and it has the advantages of high efficiency, high purity heterologous expression and low large-scale production cost, and has a high potential for industrial application.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a recombinant silk fibroin peptide SFH_R6, whose amino acid sequence is shown in SEQ ID NO.1.

[0007] The present invention also provides a gene encoding a recombinant silk fibroin peptide SFH_R6, and the nucleotide sequence thereof is shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant vector for expressing the recombinant silk fibroin peptide SFH_R6, wherein the recombinant vector comprises the above encoding gene.

[0009] In the present invention, the initial vector of the recombinant vector includes a common vector or plasmid in the art; in some embodiments of the present invention, the initial vector of the recombinant vector can be selected from a His-pet28a vector or a pPIC9K vector.

[0010] The present invention also provides a recombinant bacterium expressing the recombinant silk fibroin peptide SFH_R6, wherein the recombinant bacterium comprises the recombinant vector.

[0011] In the present invention, the starting strain of the recombinant bacteria includes a prokaryotic strain or a eukaryotic strain commonly used in the art; in some embodiments of the present invention, the starting strain of the recombinant bacteria can be selected from Escherichia coli BL21 strain or Pichia pastoris GS115 strain.

[0012] The present invention also provides a method for preparing recombinant silk fibroin peptide SFH_R6, including a prokaryotic expression method and a eukaryotic expression method;

[0013] The prokaryotic expression method comprises the steps of connecting the nucleotide sequence shown in SEQ ID NO.2 to an expression vector, constructing a recombinant vector, and then transforming it into a prokaryotic host bacterium, inducing expression, and purifying to obtain the recombinant silk protein peptide SFH_R6;

[0014] The eukaryotic expression method comprises the steps of connecting the nucleotide sequence shown in SEQ ID NO.2 to an expression vector, constructing a recombinant vector, and then transforming it into a eukaryotic host bacterium, inducing expression, and purifying to obtain the recombinant silk protein peptide SFH_R6.

[0015] Optionally, in the prokaryotic expression method, the expression vector includes a His-pet28a vector; and the prokaryotic host bacteria includes an Escherichia coli BL21 strain.

[0016] Optionally, the induced expression is induced by isopropyl-β-D-thiogalactoside; further optionally, the concentration of the isopropyl-β-D-thiogalactoside is 0.5 mM.

[0017] Optionally, the purification process includes Ni-NTA agarose crude extraction, impurity protein washing and imidazole gradient elution.

[0018] In some embodiments of the present invention, the method for crude extraction with Ni-NTA agarose is as follows: collecting the bacterial lysis supernatant after induced expression, mixing it with Ni-NTA agarose in a ratio of 4:1, equilibrated at 4°C overnight and then loaded onto the column; collecting the flow-through and repeating the loading 3-5 times.

[0019] In some embodiments of the present invention, the method for washing the impurities is: washing the column 5 times with 1-2 column volumes of Wash Buffer; the Wash Buffer: 50 mM NaH2PO4+300 mM NaCl+20 mM urea, pH 8.0.

[0020] In some embodiments of the present invention, the method of imidazole gradient elution is: eluting 5-10 times with 1 column volume of Elution Buffer containing different concentrations of imidazole; the Elution Buffer: 50mM NaH2PO4+300mM NaCl+250mM urea, pH8.0; the imidazole concentrations are 20mM, 50mM, 200mM, and 500mM, respectively.

[0021] Optionally, in the eukaryotic expression method, the expression vector includes a pPIC9K vector; and the eukaryotic host bacteria includes a Pichia pastoris GS115 strain.

[0022] Optionally, the induced expression is induced by methanol; further optionally, the final concentration of the methanol is 1%.

[0023] Optionally, the purification process includes hydrophobic chromatography, cation chromatography and anion chromatography.

[0024] In some embodiments of the present invention, the hydrophobic chromatography method is to collect the supernatant of the bacterial solution after induced expression, and use a hydrophobic elution buffer for linear elution; the hydrophobic elution buffer is obtained by mixing buffer A and buffer B; during the linear elution process, the proportion of buffer B varies between 0-100%; the number of times of the hydrophobic chromatography is 15-20 times;

[0025] The buffer A: 50 mM NaAc-HAc, 1 M (NH4)2SO4, pH 5.50;

[0026] Buffer B: 50 mM NaAc-HAc, pH 5.50;

[0027] In some embodiments of the present invention, the method of cationic chromatography is to collect the eluate after hydrophobic chromatography and use a cationic elution buffer for linear elution; the cationic elution buffer is obtained by mixing buffer C and buffer D; during the linear elution, the proportion of buffer D varies between 0-100%; the number of cationic chromatography is 10-20 times;

[0028] The buffer C: 50 mM NaAc-HAc, pH 5.0;

[0029] The buffer D: 50 mM NaAc-HAc, 0.5 M NaCl, pH 5.50.

[0030] In some embodiments of the present invention, the anion chromatography method is to collect the eluate after the cation chromatography and elute with an anion elution buffer; the number of anion chromatography is 3-5 times;

[0031] The anion elution buffer: 50 mM Tris-HAc, pH 7.2.

[0032] The present invention also provides the use of the recombinant silk fibroin peptide SFH_R6 or the recombinant silk fibroin peptide SFH_R6 prepared by the above method in the preparation of whitening products.

[0033] The present invention discloses the following technical effects:

[0034] The present invention provides a recombinant silk protein peptide SFH_R6 and a simple and efficient production method thereof. Through prokaryotic expression and eukaryotic expression, the soluble expression of the recombinant silk protein peptide is realized, and the high-purity soluble recombinant silk protein peptide SFH_R6 is obtained by nickel column affinity chromatography, hydrophobic chromatography, cation chromatography and anion chromatography technology. The recombinant silk protein peptide SFH_R6 has high purity, high tyrosinase inhibition rate and strong whitening effect. The present invention simplifies the purification process by optimizing the expression conditions, so that the purification process is simple and reproducible, and high-quality recombinant silk protein can be obtained without complicated operations. Compared with the prior art, the present invention not only reduces the production cost and reduces the technical barriers, but also greatly improves the production efficiency, effectively solves the quality control problem of natural extracted protein products, has the advantages of high efficiency, high purity heterologous expression and low large-scale production cost, has high industrial application potential, and provides strong support for the wide application of recombinant silk protein in the fields of biomedicine and cosmetic materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 The figure is the SDS-PAGE result of the induction expression and purification of the recombinant silk protein peptide SFH_R6 at 16℃; wherein, lane 1 is the bacterial solution before induction; lane 2 is the supernatant of the bacterial solution after induction; lane 3 is the washing solution of the impurities; lanes 4 and 5 are samples eluted with 100mM imidazole;

[0037] Figure 2 This is the result graph of tyrosinase inhibition rate of recombinant silk protein SFH_R6. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] The amino acid sequence of the recombinant silk fibroin peptide SFH_R6 provided by the present invention is as follows:

[0044] PWAEQDQAVLVRMLHTAVTAVTSMHGAAKATSAPVPARVRVAVPARVRVRVPVVAQV

[0045] RVTVRGWALGTAQAMVLVQARDTARVRVRVRVQVRVRVRARVLATAQAWVRAMVRVQV

[0046] AVLPSVPEPVRAQAPARVRAAVQARAPVLVRALVRARVLALALGTARGTVPASVPAMVQV

[0047] RVPARQAVRVPAAARVPDLARVRAPALARVAVRVRVLAQALATALALVPVMARVTALVLVL

[0048] AMVLVRAAVPQAVLARATTITTTKN(SEQ ID NO.1)。

[0049] The coding gene sequence of the recombinant fibroin protein peptide provided by the present invention is as follows:

[0050] ccatgggcggaacaggatcaagcggttttggtccgtatgttgcacacggcggttacagcggttacgagtatgcatggagcagcgaaag

[0051] cgacttcggcaccggttccggcgcgggtgcgggtagcggtgccggcgcgggtgcgggtgcgggtgccggtagtggcgcaggtgcgggtta

[0052] cggtgcgggggtgggcgctgggtacggcgcaggctatggtgctggtgcaggcgcgggatacggcgcgggtgcgggttcgggtacgggttc

[0053] aggtgcgggtgcgggttcgggcgcgggtgctggctacggcgcaggcgtgggtgcgggctatggtgcgggtgcaggtagcggtgctgccttc

[0054] ggtgccggagccggtgcgggcgcaggctccggcgcgggtgcgggcagcggtgcaggcgcgggctccggtgctggtgcgggctctggtgc

[0055] gggcgcgggttctggcgctggcgctgggtacggcgcggggtacggtgccggcgtcggtgccggctatggtgcaggtgcgggttccggcgc

[0056] ggcaagcggtgcgggtgccggcagcggcgcgggtgccggatctggcgcgggtgcgggctccggcgctggcgcgggtagcggtgcgggt

[0057] gcgggttctggcgcaggcgctggctacggcgctggcgttggtgccggttatggcgcgggttacggcgctggtgctggtgctggctatggtgct

[0058] ggtgcgggcagcggtgccgcaagcggtgctggcgcgggccaccaccatcaccaccactaagaattcgagctccgtcgacaagcttgcggcc

[0059] gcactcgagcaccaccaccaccaccac (SEQ ID NO. 2).

[0060] Unless otherwise specified, the biological materials or reagents involved in the present invention can be purchased through conventional channels; the operating methods involved in the present invention can be carried out according to conventional operating methods in the art unless otherwise specified.

[0061] Example 1 Prokaryotic expression and purification of recombinant silk fibroin peptide SFH_R6

[0062] The buffer used in this example is as follows:

[0063] Lysis Buffer: 50 mM NaH2PO4 + 300 mM NaCl + 10 mM urea, pH 8.0;

[0064] Wash Buffer: 50 mM NaH2PO4 + 300 mM NaCl + 20 mM urea, pH 8.0;

[0065] Elution Buffer: 50 mM NaH2PO4 + 300 mM NaCl + 250 mM urea, pH 8.0.

[0066] The nucleotide sequence shown in SEQ ID NO.2 was connected to the NdeI / EcoRI site of the expression vector plasmid His-pet28a to construct the recombinant expression vector plasmid pET28a-SFH_R6. The recombinant expression vector plasmid pET28a-SFH_R6 was transformed into the competent cell BL21 (DE3) by heat shock method, and the transformed cells were spread on the kana resistance plate, inverted and cultured at 37°C for 16h to obtain monoclonal bacteria, and the monoclonal bacteria were stored in glycerol at -80°C.

[0067] Pick a single clone and inoculate it into a 10mL LB centrifuge tube containing 50μg / mL kanamycin. Incubate it in a shaker at 37℃ and 200rpm overnight. This is called seed solution. Inoculate the seed solution into a LB shake flask containing 50μg / mL kanamycin at a ratio of 1:100. Incubate it at 37℃ and 200rpm. When the OD 600 When the pH reaches 0.6-0.8, add 0.5 mM IPTG and continue culturing at 16°C and 200 rpm for 16 h, then collect the bacteria by centrifugation at 8000 rpm for 15 min, wash 1-2 times with 0.9% NaCl solution, collect the cells by centrifugation and store at -20°C or -80°C.

[0068] Take the cells collected by centrifugation and add an appropriate amount of Lysis Buffer. Use an ultrasonic cell disruptor to disrupt the cells. The ultrasonic power is 30%, the ultrasonic treatment is 2s, the interval is 3s, the cells are disrupted for a total of 20min, and the cells are centrifuged at 4℃5000g for 40min. The supernatant is collected and purified through a 0.45μm filter membrane. Mix the bacterial lysate supernatant and the nickel column (Ni-NTA) in a volume ratio of 4:1, and shake slowly on a horizontal shaker at 4℃ overnight. The mixture of the bacterial lysate supernatant and the nickel column is loaded into an empty column tube. Open the lid at the bottom of the purification column tube, let the liquid in the column flow out under gravity, collect the flow-through and repeat the column 5 times to fully bind the target protein. Then balance the Ni affinity chromatography medium with Wash Buffer, add 1-2 column volumes of Wash Buffer each time, wash the column 5 times, and collect the impurity protein washing liquid. Finally, the cells were eluted with different concentrations of imidazole (20mM, 50mM, 200mM, 500mM) in Elution Buffer, and the eluents were collected. The 200mM imidazole eluent was used for sample preparation and gel running verification. 10% SDS PAGE electrophoresis was performed, and the cells were stained with instant blue dye for 15 minutes and decolorized on a shaking table with clean water for more than 1 hour. The images were scanned by a gel scanning imaging system (Tanon, MINI Space 1000). The results showed that the target protein SFH_R6 (such as Figure 1 shown).

[0069] Example 2 Eukaryotic expression and purification of recombinant silk fibroin peptide SFH_R6

[0070] The buffer used in this example is as follows:

[0071] Buffer A: 50 mM NaAc-HAc, 1 M (NH4)2SO4, pH 5.50;

[0072] Buffer B: 50 mM NaAc-HAc, pH 5.50;

[0073] Buffer C: 50 mM NaAc-HAc, pH 5.0;

[0074] Buffer D: 50 mM NaAc-HAc, 0.5 M NaCl, pH 5.50;

[0075] Anion elution buffer: 50 mM Tris-HAc, pH 7.2.

[0076] The nucleotide sequence shown in SEQ ID NO.2 was connected to the NdeI / EcoRI position of the expression vector plasmid pPIC9K to construct the recombinant expression vector plasmid pPIC9K-SFH_R6. The recombinant expression vector plasmid pPIC9K-SFH_R6 was transformed into Pichia pastoris GS115 by electroporation, and the monoclonal cells were harvested after cultivation.

[0077] Place the single clone in a 250 mL conical flask containing 25 mL of BMGY medium and culture overnight, which is recorded as the seed solution. Inoculate the seed solution into 1 L of BMGY medium at a ratio of 1:50 and continue to culture at 30°C and 220 r / min until the OD 600 About 0.6-0.8, centrifuged at 5000r / min for 5min, discarded the supernatant, collected the cells and resuspended them in 1L BMMY culture medium, and added methanol with a final concentration of 1%. Continued to culture at 30℃ and 220r / min for 72h, and methanol was added every 24h to a final concentration of 1%. The supernatant culture medium was collected for subsequent separation and purification of the recombinant silk protein peptide SFH_R6.

[0078] The supernatant culture fluid is loaded onto a chromatography column loaded with a hydrophobic resin, and linear elution is performed with 1 column volume of a hydrophobic elution buffer each time, and the column is washed 15 times in total to obtain a first eluate. The hydrophobic elution buffer is obtained by mixing buffer A with buffer B, and the proportion of buffer B in the hydrophobic elution buffer varies between 0-100%.

[0079] The first eluate is loaded onto a chromatography column loaded with a cationic resin, and cation elution is performed with 1 column volume of a cationic elution buffer each time, and the column is washed 15 times in total to obtain a cationic eluate. The cationic elution buffer is obtained by mixing buffer C and buffer D, and the proportion of buffer D in the cationic elution buffer varies between 0-100%.

[0080] The cationic eluate was loaded onto a chromatography column containing an anionic resin, and anionic chromatography was performed using 1 column volume of anionic elution buffer each time. The column was washed 5 times, and the eluate was collected and freeze-dried through ultrafiltration with a 0.45 μm filter membrane to obtain the purified recombinant silk protein peptide SFH_R6 freeze-dried product.

[0081] Test example: Detection of tyrosinase inhibition rate of recombinant silk protein SFH_R6

[0082] Tyrosinase activity inhibition test was performed using L-tyrosine as substrate. The total reaction system was 200 μL, including 40 μL sample solution, 140 μL substrate solution and 20 μL enzyme solution. The substrate solution was prepared with PBS to a final concentration of 1.5 mmol / L. The final mass concentration of the enzyme solution was 0.33 U / μL. The sample solution was prepared with PBS, and the recombinant silk protein peptide SFH_R6 purified in Example 2 was diluted to a concentration of 0.25 mg·mL -1 , 0.5mg·mL -1 , 0.75mg·mL -1 , 1mg·mL -1 Gradient sample solution. During the assay, the solution was added to a 96-well plate according to the dosage in Table 1 and shaken at 25°C for 20 minutes. The absorbance value was then obtained at 475nm using an ELISA reader. Ascorbic acid (Vitamin C, Vc) was used as a positive control, and 3 tests were performed in parallel in each group.

[0083] Table 1 Reaction system

[0084]

[0085] The tyrosinase inhibition rate calculation formula is as follows:

[0086] Inhibition rate (%) = (1-ODsample / ODcontrol) × 100

[0087] The results are as follows Figure 2As shown, in the recombinant silk protein SFH_R6 experimental group, all concentrations of recombinant silk protein peptide SFH_R6 have tyrosinase inhibitory effects, and the inhibitory activity rate increases with the increase of its concentration. Among them, when the concentration of silk protein SFH_R6 is 1.0 mg / mL, its inhibition rate on tyrosinase activity can reach 99%, even higher than that of the control group. This result shows that the recombinant silk protein peptide provided by the present invention has the effect of inhibiting tyrosinase activity, has the ability to whiten skin, and is a potential raw material for whitening cosmetics.

[0088] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A recombinant silk fibroin peptide SFH_R6, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding a recombinant silk fibroin peptide SFH_R6, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant vector expressing recombinant silk fibroin peptide SFH_R6, characterized in that: The recombinant vector contains the coding gene according to claim 2.

4. A recombinant bacterium expressing recombinant silk fibroin peptide SFH_R6, characterized in that: The recombinant bacteria comprises the recombinant vector according to claim 3.

5. A method for preparing recombinant silk fibroin peptide SFH_R6, characterized in that: Including prokaryotic expression methods and eukaryotic expression methods; The prokaryotic expression method comprises the steps of connecting the nucleotide sequence shown in SEQ ID NO.2 to an expression vector, constructing a recombinant vector, and then transforming it into a prokaryotic host bacterium, inducing expression, and purifying to obtain the recombinant silk protein peptide SFH_R6; The eukaryotic expression method comprises the steps of connecting the nucleotide sequence shown in SEQ ID NO.2 to an expression vector, constructing a recombinant vector, and then transforming it into a eukaryotic host bacterium, inducing expression, and purifying to obtain the recombinant silk protein peptide SFH_R6.

6. The method according to claim 5, characterized in that In the prokaryotic expression method, the expression vector includes a His-pet28a vector; and the prokaryotic host bacteria includes an Escherichia coli BL21 strain.

7. The method according to claim 5, characterized in that The induced expression is induced by isopropyl-β-D-thiogalactoside; The purification process includes Ni-NTA agarose crude extraction, impurity protein washing and imidazole gradient elution.

8. The method according to claim 5, characterized in that In the eukaryotic expression method, the expression vector includes a pPIC9K vector; and the eukaryotic host bacteria includes a Pichia pastoris GS115 strain.

9. The method according to claim 5, characterized in that The induced expression is induced by methanol; The purification process includes hydrophobic chromatography, cation exchange chromatography and anion exchange chromatography.

10. Use of the recombinant silk fibroin peptide SFH_R6 according to claim 1 or the recombinant silk fibroin peptide SFH_R6 prepared by the method according to any one of claims 5 to 9 in the preparation of whitening products.

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