Recombinant fibronectin and preparation method and application thereof

Abstract

The application belongs to the technical field of biology, and particularly relates to a recombinant fibronectin as well as a preparation method and application thereof. The preparation method of the recombinant fibronectin provided by the application comprises the following steps: constructing a recombinant plasmid containing an optimized nucleotide sequence (SEQ ID NO. 2), transforming Escherichia coli and inducing expression; after the bacterial body is resuspended by a buffer and homogenized by high pressure, alkaline protease is added for one-step enzymolysis, and after inactivation, the precipitate is obtained by centrifugation, washed by NaCl and dissolved by acetic acid, so that a recombinant fibronectin stock solution is obtained, and the amino acid sequence of the recombinant fibronectin (rhFNb) is shown as SEQ ID NO. 1. The preparation method provided by the application does not need chromatography purification, and the refining of the recombinant fibronectin can be completed within 15 minutes, so that the production cycle is effectively shortened while the product purity is ensured to be greater than or equal to 95% and the adhesion activity is high.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a recombinant fibronectin as well as a preparation method and application thereof. BACKGROUND

[0002] Fibronectin (FN) is a large molecular glycoprotein, which is widely present in the extracellular matrix (ECM) and plasma of vertebrates, and plays a crucial role in the interaction between cells and extracellular matrix, and is one of the key molecules in cell biology and developmental biology. Fibronectin is composed of repeated functional modules, has a complex molecular structure, and exists in three types of modules, mainly including 12 type I modules, 2 type II modules and 15 type III modules, and contains variable spliceable regions (ED-A, ED-B, IIICS), which endow fibronectin with rich functional diversity and tissue specificity. Through its specific domains, fibronectin binds to integrin receptors on the cell surface on the one hand, and binds to other components (such as collagen, fibrin, heparin, etc.) in the extracellular matrix on the other hand, and plays an indispensable role in key biological processes such as cell adhesion, spreading, migration, proliferation, differentiation, embryonic development, tissue repair, wound healing, etc. Therefore, fibronectin is a key molecule in the fields of cell biology, developmental biology, regenerative medicine and wound repair.

[0003] In recent years, with the development of tissue engineering and regenerative medicine, the research and application of extracellular matrix have been highly valued, and the application potential of fibronectin and its functional fragments in promoting cell behavior and accelerating tissue or wound repair has attracted great attention. In order to obtain fibronectin or its fragments with specific biological activity, the existing technology generally uses a heterologous expression system (such as Escherichia coli, yeast, mammalian cells) for recombinant production. The corresponding products are mainly used in the technical field of promoting tissue or wound repair. However, there are still significant shortcomings and limitations in the extraction and purification technology of existing fibronectin active fragments.

[0004] Fibronectin has a large molecular weight and a complex structure (multi-domain, dimeric form), and there may be host protein contamination, misfolding or aggregate formation in the heterologous expression system, which leads to very tedious and inefficient subsequent purification steps (such as chromatographic separation), resulting in great difficulty in purification and separation. The complex purification process requires expensive chromatographic media and equipment, consumes a large amount of manpower and material resources, and the yield of target protein is often not high, ultimately leading to high unit cost of products. The production cycle needs to go through gene cloning, expression, multi-step purification and activity verification, and the whole process is time-consuming and difficult to meet the rapid and flexible production demand.

[0005] In summary, the prior art in the application of fibronectin is faced with problems including difficulty in purification and separation, high cost, long period, etc. Therefore, it is necessary to study a method with high efficiency, low cost and short period, which can realize rapid and effective separation and purification of fibronectin or specific functional fragments (target proteins) with high biological activity, so as to overcome the bottleneck of the existing heterologous expression technology and promote the practical transformation in biomedical applications. SUMMARY

[0006] In order to achieve the above-mentioned purpose, the fibronectin fragment containing the high-activity region of fibronectin is selected, and a one-step enzymatic hydrolysis method is used for purification, which is simple and efficient, and the obtained recombinant fibronectin has the characteristics of simple production process and high adhesion activity.

[0007] The first aspect of the present application provides a rapid preparation method of the recombinant fibronectin, which specifically comprises the following steps:

[0008] S1, chemically synthesizing the sequence shown in SEQ ID NO. 2, inserting the sequence shown in SEQ ID NO. 2 into a vector by enzyme digestion to obtain a recombinant vector;

[0009] S2, transforming the recombinant vector in step S1 into Escherichia coli BL21 DE3 and inducing expression;

[0010] S3, collecting the bacterial body after the induction expression in step S2 is completed, mixing uniformly after adding a buffer solution to obtain a bacterial suspension, then homogenizing and incubating to obtain an incubation product;

[0011] S4, adding alkaline protease to the incubation product in step S3, mixing uniformly, reacting to obtain a reaction liquid, then adding acid to adjust the pH to 2.0~5.0 to terminate the reaction;

[0012] S5, centrifuging the product after the reaction is terminated in step S4, taking the precipitate, washing and dissolving, and then obtaining the product.

[0013] Preferably, the enzyme digestion in step S1 is double enzyme digestion, and the restriction endonuclease used in the double enzyme digestion is NdeI and BamHI;

[0014] Preferably, the culture temperature of the induction expression in step S2 is 16℃~42℃; the inoculation amount is 0.01%~10%; the inducer is added when the OD600 of the bacterial body is between 0.1~0.5; the inducer is IPTG; the final concentration of the IPTG is 0.1~1mM; and the induction time is 2~10h;

[0015] Preferably, the culture temperature of the induction expression is 37℃; the inoculation amount is 2%; the inducer is added when the OD600 of the bacterial body is 0.4; the inducer is IPTG; the final concentration of the IPTG is 0.8mM; and the induction time is 4h;

[0016] Preferably, the homogenization in step S3 is adding the bacterial suspension in step S3 into a high-pressure homogenizer, and repeating the homogenization 2-4 times under the conditions of 15-20℃ and 400-600bar; and incubating the intermediate product after homogenization under the conditions of 15-20℃ for 5-15min.

[0017] Preferably, the buffer in step S3 is a phosphate buffer or a Tris buffer.

[0018] Preferably, the buffer in step S3 is a phosphate buffer with pH=7.5 and a concentration of 50mM, or a Tris buffer with pH=7.5 and a concentration of 20mM.

[0019] Preferably, the alkaline protease is added in a ratio of 5-10U per gram of wet weight of the starting bacterial body in step S4.

[0020] Preferably, the reaction temperature in step S4 is 15-20℃, and the reaction time is 10-20min.

[0021] Preferably, the centrifugation in step S5 is under the conditions of 7000-9000g for 5-15min.

[0022] Preferably, the washing in step S5 is washing the precipitate in step S5 with 0.5M NaCl or 1M NaCl twice, and the dissolving is with 1% acetic acid.

[0023] Preferably, the mass ratio of the bacterial body to the buffer in step S3 is 1:5-8, and the mass of the bacterial body is the wet weight.

[0024] Preferably, the amino acid sequence of the recombinant fibronectin is shown in SEQ ID NO.1. The present application refers to the fibronectin amino acid sequence in the UNIPROT database (P02751, online website: FN1 - Fibronectin - Homosapiens (Human) | UniProtKB | UniProt), selects the collagen-binding domain, combines with the RGD sequence, and adds a histidine signature tag, and is named rhFNb.

[0025] The SEQ ID NO.1 is specifically:

[0026] MHHHHHHRGDCVTDSGVVYSVGMQWLKTQGNKQMLCTCLGNGVSCQETAVTQTYGGNSNGEPCVLPFTYNGRTFYSCTTEGRQDGHLWCSTTSNYEQDQKYSFCTDHTVLVQTRGGNSNGALCHFPFLYNNHNYTDCTSEGRRDNMKWCGTTQNYDADQKFGFCPMAAHEEICTTNEGVMYRIGDQWDKQHDMGHMMRCTCVGNGRGEWTCIAYSQLRDQCIVDDITYNVNDTFHKRHEEGHMLNCTCFGQGRGRWKCDPVDQCQDSETGTFYQIGDSWEKYVHGVRYQCYCYGRGIGEWHCQPLQTYPSSRGDHHHHHH*. Wherein, * corresponds to a stop codon.

[0027] Preferably, the nucleic acid sequence encoding the recombinant fibronectin is as shown in SEQ ID NO. 2, specifically as follows:

[0028] CATATGCATCATCATCATCATCATCGCGGCGATTGCGTGACCGATAGCGGCGTGGTGTATAGCGTGGGCATGCAGTGGCTGAAAACCCAGGGCAACAAACAGATGCTGTGCACCTGCCTGGGCAACGGCGTGAGCTGCCAGGAAACCGCGGTGACCCAGACCTATGGCGGCAACAGCAACGGCGAACCGTGCGTGCTGCCGTTTACCTATAACGGCCGCACCTTTTATAGCTGCACCACCGAAGGCCGCCAGGATGGCCATCTGTGGTGCAGCACCACCAGCAACTATGAACAGGATCAGAAATATAGCTTTTGCACCGATCATACCGTGCTGGTGCAGACCCGCGGCGGCAACAGCAACGGCGCGCTGTGCCATTTTCCGTTTCTGTATAACAACCATAACTATACCGATTGCACCAGCGAAGGCCGCCGCGATAACATGAAATGGTGCGGCACCACCCAGAACTATGATGCGGATCAGAAATTTGGCTTTTGCCCGATGGCGGCGCATGAAGAAATTTGCACCACCAACGAAGGCGTGATGTATCGCATTGGCGATCAGTGGGATAAACAGCATGATATGGGCCACATGATGCGCTGCACCTGCGTGGGCAACGGCCGCGGCGAATGGACCTGCATTGCGTATAGCCAGCTGCGCGATCAGTGCATTGTGGATGATATTACCTATAACGTGAACGATACCTTTCATAAACGCCATGAAGAAGGCCACATGCTGAACTGCACCTGCTTTGGCCAGGGCCGCGGCCGCTGGAAATGCGATCCGGTGGATCAGTGCCAGGATAGCGAAACCGGCACCTTTTATCAGATTGGCGATAGCTGGGAAAAATATGTGCATGGCGTGCGCTATCAGTGCTATTGCTATGGCCGCGGCATTGGCGAATGGCATTGCCAGCCGCTGCAGACCTATCCGAGCAGCCGCGGCGATCATCATCATCATCATCATTGA TGAGGATCC In the above sequence, the underlined portion is a restriction enzyme site.

[0029] The present application obtains the E. coli preferred codon optimized sequence of the nucleic acid sequence of the recombinant fibronectin by codon optimization.

[0030] Preferably, the E. coli is BL21 DE3 E. coli.

[0031] Another aspect of the present application provides the use of the rapid preparation method in the preparation of fibronectin-containing products.

[0032] Preferably, the fibronectin-containing product includes a cell culture substrate, a medical wound repair dressing, a tissue engineering scaffold, a repair skin care product, and a biocompatible coating material.

[0033] The electrophoretic purity of the fibronectin purified by the rapid preparation method provided by the present application is above 95%. The purity detection method used is to perform gray scale scanning on the electrophoresis diagram by using ImageJ.

[0034] Compared with the prior art, the present application has the following beneficial effects:

[0035] (1) The rapid preparation method of the present application can quickly obtain recombinant fibronectin with high purity through simple steps in purification, and the product has high adhesion activity.

[0036] (2) Based on natural fibronectin, the present application selects fragments for combination and heterologous expression, and the specific spatial structure has certain protease tolerance, which is suitable for the rapid preparation method of the present application.

[0037] (3) The recombinant fibronectin amino acid sequence provided by the present application is suitable for various expression hosts, and has a wide range of uses. The expression host can be E. coli, Bacillus subtilis, Bacillus licheniformis, tobacco mosaic virus, yeast, mammalian cells, etc., which is beneficial to experimental or industrial production.

[0038] (4) The rapid preparation method provided by the present application realizes the purification of recombinant fibronectin by adding alkaline protease through one-step enzymolysis, which is simple and efficient, does not require additional equipment, has low production cost, and the final purity can reach above 95%. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 SDS-PAGE diagram of supernatant under different enzymolysis conditions;

[0040] Figure 2 Wb verification diagram of enzymolysis product;

[0041] Figure 3 SDS-PAGE diagram of the prepared recombinant fibronectin stock solution;

[0042] Figure 4 Figure 2 shows the cell adhesion activity of different volume percentages of rhFNb crude solution purified by one-step enzymatic method (protein concentration of crude solution 100 μg / mL). DETAILED DESCRIPTION

[0043] The application will be further described in detail below in conjunction with the examples. The following examples are merely illustrative, and are not intended to limit the present application.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The materials, methods, and examples provided herein are illustrative only and not intended to be limiting.

[0045] Example 1 Design and expression verification of recombinant fibronectin

[0046] 1. Sequence design: Referring to the fibronectin amino acid sequence (P02751, online address: FN1 - Fibronectin - Homo sapiens (Human) | UniProtKB | UniProt) in the UNIPROT database, the collagen-binding domain was selected and combined with the RGD sequence, and a histidine signature tag was added, named rhFNb. The amino acid sequence of rhFNb is shown in SEQ ID NO. 2:

[0047] MHHHHHHRGDCVTDSGVVYSVGMQWLKTQGNKQMLCTCLGNGVSCQETAVTQTYGGNSNGEPCVLPFTYNGRTFYSCTTEGRQDGHLWCSTTSNYEQDQKYSFCTDHTVLVQTRGGNSNGALCHFPFLYNNHNYTDCTSEGRRDNMKWCGTTQNYDADQKFGFCPMAAHEEICTTNEGVMYRIGDQWDKQHDMGHMMRCTCVGNGRGEWTCIAYSQLRDQCIVDDITYNVNDTFHKRHEEGHMLNCTCFGQGRGRWKCDPVDQCQDSETGTFYQIGDSWEKYVHGVRYQCYCYGRGIGEWHCQPLQTYPSSRGDHHHHHH*.

[0048] 2. Codon optimization: According to the preference of E. coli, the codons are optimized, and the nucleotide sequence obtained after optimization is shown in SEQ ID NO. 2, wherein the underlined part is the enzyme cutting site:

[0049] CATATGCATCATCATCATCATCATCGCGGCGATTGCGTGACCGATAGCGGCGTGGTGTATAGCGTGGGCATGCAGTGGCTGAAAACCCAGGGCAACAAACAGATGCTGTGCACCTGCCTGGGCAACGGCGTGAGCTGCCAGGAAACCGCGGTGACCCAGACCTATGGCGGCAACAGCAACGGCGAACCGTGCGTGCTGCCGTTTACCTATAACGGCCGCACCTTTTATAGCTGCACCACCGAAGGCCGCCAGGATGGCCATCTGTGGTGCAGCACCACCAGCAACTATGAACAGGATCAGAAATATAGCTTTTGCACCGATCATACCGTGCTGGTGCAGACCCGCGGCGGCAACAGCAACGGCGCGCTGTGCCATTTTCCGTTTCTGTATAACAACCATAACTATACCGATTGCACCAGCGAAGGCCGCCGCGATAACATGAAATGGTGCGGCACCACCCAGAACTATGATGCGGATCAGAAATTTGGCTTTTGCCCGATGGCGGCGCATGAAGAAATTTGCACCACCAACGAAGGCGTGATGTATCGCATTGGCGATCAGTGGGATAAACAGCATGATATGGGCCACATGATGCGCTGCACCTGCGTGGGCAACGGCCGCGGCGAATGGACCTGCATTGCGTATAGCCAGCTGCGCGATCAGTGCATTGTGGATGATATTACCTATAACGTGAACGATACCTTTCATAAACGCCATGAAGAAGGCCACATGCTGAACTGCACCTGCTTTGGCCAGGGCCGCGGCCGCTGGAAATGCGATCCGGTGGATCAGTGCCAGGATAGCGAAACCGGCACCTTTTATCAGATTGGCGATAGCTGGGAAAAATATGTGCATGGCGTGCGCTATCAGTGCTATTGCTATGGCCGCGGCATTGGCGAATGGCATTGCCAGCCGCTGCAGACCTATCCGAGCAGCCGCGGCGATCATCATCATCATCATCATTGA TGAGGATCC .

[0050] 3. Preparation of recombinant fibronectin: The above gene sequence was entrusted to GenScript to synthesize, and was ligated into pET3C vector by NdeI and BamHI double enzyme digestion to obtain pET3C-rhFNb. pET3C-rhFNb was transformed into E. coli BL21 DE3 for induced expression. The culture temperature for induced expression was 37°C; the inoculation amount was 2%; the inducer IPTG was added when the OD600 of the bacterial cells was 0.4; the final concentration of IPTG was 0.8 mM; and the induction time was 4 h. The expression product was further verified by Western blotting experiment (detection of histidine tag monoclonal antibody), and the specific operation method of Western blotting experiment was referred to the fourth edition of Molecular Cloning Experiment Guide, Science Press.

[0051] The bacterial cells after induced expression were mixed with buffer solution pH = 7.5 and 50 mM phosphate buffer solution at a ratio of 1:5 (wet weight of bacterial cells: mass of buffer solution) to obtain a bacterial suspension. Then the bacterial suspension was added to a high-pressure homogenizer, and was repeatedly homogenized at 20°C and 500 bar for 3 times. The intermediate product after homogenization was incubated at 16°C for 10 min. At the end of the incubation, 5 U of alkaline protease (Novozymes Savinase Ultra 16L CAS: 87199-17-5) was added per gram of wet weight of the starting bacterial cells, and then 7.5 U of alkaline protease was added per gram of wet weight of the starting bacterial cells after the incubation, and the mixture was incubated at 16°C for 15 min to obtain a reaction solution. Then acetic acid was added to the reaction solution to adjust the pH to 3.5, inactivate the alkaline protease, and terminate the reaction. Then the reaction solution was centrifuged at 8000g for 10 min to remove the supernatant and obtain a precipitate. The sample after incubation was centrifuged to obtain the supernatant sample before enzymolysis.

[0052] The specific steps of Western blotting experiment are as follows:

[0053] 1. The supernatant sample and the precipitate sample after the enzymolysis reaction were subjected to SDS-PAGE electrophoresis to obtain the target protein.

[0054] 2. The buffer solution was prepared: transfer solution (5.8 g glycine, 11.6 g Tris-base, 0.74 g SDS, 400 ml methanol, and ddH2O to 2 L), 1x TBST (20 ml 1M Tris-HCl, 17.6 g NaCl, 2 ml Tween-20, and ddH2O to 2 L).

[0055] 3. Transfer: constant current 260 mA, transfer for 70 min.

[0056] 4. Blocking: after the transfer was completed, the blocking solution was used for blocking at room temperature for 1 h.

[0057] 5. Wash: After the blocking, wash the membrane with 1x TBST for 10 min, 60 rpm, 3 times.

[0058] 6. Antibody incubation: Incubate with HRP-conjugated 6*His, His-Tag Monoclonal antibody directly for 1.5 h at room temperature. No secondary antibody is needed after the incubation. The membrane can be directly developed after washing.

[0059] 7. After the incubation, wash the membrane with 1x TBST for 10 min, 60 rpm, 5 times.

[0060] 8. Development.

[0061] The results are shown in Figure 2 . Figure 2 Lane 1 is the supernatant before enzyme digestion; lane 2 is the negative control; lane 3 is the precipitate after enzyme digestion; lane 4 is the supernatant after enzyme digestion. It can be seen that the target protein appears in the precipitate after enzyme digestion, and the target protein is almost not contained in the supernatant after enzyme digestion. Samples with different enzyme digestion times were prepared using the above bacteria and the same method to verify the enzyme digestion effect, and the results are shown in Figure 1 . Lanes 1-6 correspond to 10 min, 20 min, 30 min, 40 min, 50 min of enzyme digestion time, respectively.

[0062] Example 2 A recombinant fibronectin

[0063] A recombinant fibronectin, and the specific purification steps are as follows:

[0064] The bacteria induced to express in Example 1 were collected, and the collected bacteria and buffer solution pH = 7.5, concentration 50 mM phosphate buffer solution were mixed at a mass ratio of 1:5 of the wet weight of the bacteria to the buffer solution to obtain a bacterial suspension. Then the bacterial suspension was added to a high-pressure homogenizer, and homogenized at 20°C and 600 bar for 4 times. The intermediate product after homogenization was incubated at 16°C for 10 min. Alkaline protease (Merck P5380, CAS: 9014-01-1) was added at a ratio of 5 U per gram of wet weight of the starting bacteria after the incubation, and mixed after the addition of the alkaline protease. Then the reaction was carried out at 16°C for 15 min to obtain a reaction solution. Then acid was added to the reaction solution to adjust the pH to 2.0 to inactivate the alkaline protease and terminate the reaction. Then the supernatant was removed by centrifugation at 7000g for 10 min to obtain a precipitate. The precipitate was washed with 0.5M NaCl twice, and then dissolved with 1% acetic acid to obtain a recombinant fibronectin stock solution.

[0065] Figure 3Lane 1-5 correspond to the final product of enzymatic hydrolysis time of 50 min, 40 min, 30 min, 20 min, and 10 min, respectively. The electrophoretic results of fibronectin purified in this example are shown in Figure 3 The purity of fibronectin in the sample was calculated by ImageJ gray scale scanning, and the result was that the purity was above 95%.

[0066] Example 3: A recombinant fibronectin

[0067] A recombinant fibronectin, and the specific purification steps are as follows:

[0068] The bacteria induced for expression in Example 1 were collected, and the collected bacteria were mixed with Tris buffer solution with a pH of 7.5 and a concentration of 20 mM at a ratio of 1:8 of the wet weight of the bacteria to the mass of the buffer solution to obtain a bacterial suspension. Then the bacterial suspension was added to a high-pressure homogenizer, and repeated homogenization was performed at 15°C and 400 bar for 3 times. The intermediate product after homogenization was incubated at 18°C for 5 min. Alkaline protease was added at a ratio of 10 U per gram of the wet weight of the starting bacteria after the incubation was completed, and the mixture was incubated at 15°C for 20 min to obtain a reaction solution. Then acid was added to the reaction solution to adjust the pH to 5.0, inactivate the alkaline protease, and terminate the reaction. Then centrifugation was performed at 9000g for 10 min, and the supernatant was removed to obtain a precipitate. The precipitate was washed with 1M NaCl twice, and then dissolved with 1% acetic acid to obtain a recombinant fibronectin stock solution.

[0069] The purity of the purified fibronectin was above 95%.

[0070] Example 4: A recombinant fibronectin

[0071] A recombinant fibronectin, which is basically the same as Example 2, the only difference is that the incubation time is 20 min. The product obtained is shown in Figure 3 Lane 4.

[0072] Example 5: A recombinant fibronectin

[0073] A recombinant fibronectin, which is basically the same as Example 2, the only difference is that the incubation time is 30 min. The product obtained is shown in Figure 3 Lane 3.

[0074] Example 6: A recombinant fibronectin

[0075] A recombinant fibronectin, which is basically the same as Example 2, the only difference is that the incubation time is 40 min. The product obtained is shown in Figure 3 Lane 2.

[0076] Example 7: A recombinant fibronectin

[0077] A recombinant fibronectin, essentially the same as in Example 2, except that the incubation time was 50 min. The resulting product is as follows: Figure 4 As shown in lane 1.

[0078] Experimental Example 1: Product Activity Verification

[0079] The adhesion activity of the recombinant fibronectin prepared in Example 2 was determined using the method in Appendix B of YY / T 1849-2022. The results are shown below. Figure 4 .Depend on ​ It can be seen that the recombinant fibronectin obtained by one-step enzymatic hydrolysis in Example 2 has good adhesion activity. As the concentration increases exponentially, the cell adhesion rate begins to decrease when the volume percentage of the recombinant fibronectin stock solution rises to 12.5%. This is because the stock solution contains acetic acid, and excessive acetic acid content will inhibit cell adhesion activity.

[0080] Comparative Example 1

[0081] The fibronectin mutant was prepared using the method disclosed in Chinese Patent CN117466992A. The specific steps are as follows:

[0082] The cell lysate supernatant of the fibronectin mutant was diluted with water to a conductivity of 1 ms / cm and then purified by chromatography. First, a cation exchange column (SP Sepharose FF) was used for flow-through (during which some non-target proteins were adsorbed onto the packing material). The resulting target protein flow-through was then washed with an anion exchange column (Q Sepharose FF) for adsorption and high-salt elution (20 mM sodium phosphate buffer, containing 300 mM sodium chloride, pH 7.0) to obtain the high-purity target protein. Finally, the high-purity target protein was subjected to tangential flow filtration (using a 5 KD filter membrane) with buffer exchange (20 mM sodium phosphate buffer, pH 7.0) to obtain the fibronectin mutant. The electrophoresis image of this fibronectin mutant is shown in patent CN117466992A.

[0083] Comparative Example 2

[0084] Recombinant human fibronectin was prepared using the method disclosed in Chinese Patent CN119798419A. The specific steps are as follows:

[0085] 1. Obtain the protein extract:

[0086] Each 1g of engineered bacterial sludge obtained in Example 1 was resuspended in 10mL of Lysis Buffer-1, crushed 2-3 times under 800Pa pressure, centrifuged at 4℃ and 12000rpm for 30min, the supernatant was collected and filtered through a 0.22μm filter membrane to obtain the total protein extract.

[0087] 2. Nickel column purification:

[0088] (1) Buffer preparation:

[0089] Lysis Buffer-1: 50 mM Tris-HCl, 150 mM NaCl and 50 mM imidazole, pH 8.0 adjusted with NaOH solution, sterilized by filtration through 0.22 μm membrane;

[0090] Wash Buffer-1: 50 mM Tris-HCl, 150 mM NaCl and 50 mM imidazole, pH 8.0 adjusted with NaOH solution, sterilized by filtration through 0.22 μm membrane;

[0091] Wash Buffer-2: 50 mM Tris-HCl, 150 mM NaCl and 100 mM imidazole, pH 8.0 adjusted with NaOH solution, sterilized by filtration through 0.22 μm membrane;

[0092] Wash Buffer-3: 50 mM Tris-HCl, 150 mM NaCl and 150 mM imidazole, pH 8.0 adjusted with NaOH solution, sterilized by filtration through 0.22 μm membrane;

[0093] Elution Buffer-1: 50 mM Tris-HCl, 150 mM NaCl and 200 mM imidazole, pH 8.0 adjusted with NaOH solution, sterilized by filtration through 0.22 μm membrane;

[0094] Elution Buffer-2: 50 mM Tris-HCl, 150 mM NaCl and 500 mM imidazole, pH 8.0 adjusted with NaOH solution, sterilized by filtration through 0.22 μm membrane;

[0095] (2) Equilibrate the nickel column with 10 column volumes of Lysis Buffer-1;

[0096] (3) Load the total protein extract from step 1 onto the nickel column, collect the flow-through;

[0097] (4) Equilibrate the nickel column with 10 column volumes of Lysis Buffer-1;

[0098] (5) Add 5 column volumes of Wash Buffer-1, 5 column volumes of Wash Buffer-2, 5 column volumes of Wash Buffer-3, 5 column volumes of Elution Buffer-1 and 5 column volumes of Elution Buffer-2 sequentially, collect the washes and eluate fractionally;

[0099] (6) The eluate is collected and concentrated, desalted to obtain a desalted solution, and then concentrated again to obtain a re-concentrated solution. The re-concentrated waste liquid is collected;

[0100] SDS-PAGE electrophoresis analysis is performed, and the purity is >90%.

[0101] 3. Anion column purification:

[0102] (1) Buffer preparation:

[0103] Lysis Buffer-2: 50mM Tris-HCl, 50mM NaCl, adjusted to pH 8.0 with NaOH solution, and filtered through a 0.22μm filter to remove bacteria;

[0104] Wash Buffer-3: 50mM Tris-HCl and 50mM NaCl, adjusted to pH 8.0 with NaOH solution, and filtered through a 0.22μm filter to remove bacteria;

[0105] Wash Buffer-4: 50mM Tris-HCl and 200mM NaCl, adjusted to pH 8.0 with NaOH solution, and filtered through a 0.22μm filter to remove bacteria;

[0106] Elution Buffer-3: 50mM Tris-HCl and 500mM NaCl, adjusted to pH 8.0 with NaOH solution, and filtered through a 0.22μm filter to remove bacteria;

[0107] (2) Equilibrate the nickel column with 10 column volumes of Lysis Buffer-2;

[0108] (3) Load the re-concentrated solution obtained in step 1 into the nickel column, and collect the flow-through liquid 2;

[0109] (4) Equilibrate the nickel column with 10 column volumes of Lysis Buffer-2;

[0110] (5) Add 5 column volumes of Wash Buffer-3, 5 column volumes of Wash Buffer-4, and 5 column volumes of Elution Buffer-3 in sequence, and collect the eluate and the eluate;

[0111] (6) Concentrate the eluate, desalt to obtain a desalted solution, and then concentrate again to obtain a re-concentrated solution. The re-concentrated waste liquid is collected;

[0112] SDS-PAGE electrophoresis analysis is performed, and the purity is >95%. The electropherogram of the fibronectin mutant is shown in patent CN119798419A.

[0113] As can be seen from the comparison of Examples 2-3 and Comparative Examples 1-2, the recombinant fibronectin and the preparation method of the recombinant fibronectin provided by the present application can be used to quickly and simply prepare a recombinant fibronectin stock solution with a purity of >95%, which can meet the rapid and flexible requirements in research and production.

Claims

1. A method for rapid production of recombinant fibronectin, characterized by, Specifically comprising the following steps: S1, chemically synthesizing the sequence shown in SEQ ID NO. 2, inserting the sequence shown in SEQ ID NO. 2 into a vector by enzyme digestion to obtain a recombinant vector; S2, transforming the recombinant vector of step S1 into E. coli and inducing expression; S3, collecting the bacterial cells after the expression induced in step S2, adding a buffer to mix, obtaining a bacterial suspension, then homogenizing and incubating to obtain an incubation product; the buffer is a phosphate buffer or a Tris buffer; S4, adding alkaline protease into the incubation product of step S3, mixing, reacting, obtaining a reaction solution, and then adding acid to adjust the pH to 2.0 5.0, terminating the reaction; the alkaline protease is added at a ratio of 5-10 U per gram of wet weight of the starting bacteria; the reaction temperature is 15-20°C, and the reaction time is 10-20 min. S5, centrifuging the product after the reaction of step S4 is terminated, the centrifugation conditions are 7000 ~ 9000g, 5 ~ 15min, taking the precipitate, washing, dissolving, and obtaining the product.

2. The rapid production method according to claim 1, characterized by, The enzyme digestion of step S1 is double enzyme digestion, and the restriction endonucleases used in the double enzyme digestion are NdeI and BamHI.

3. The rapid production method according to claim 1, characterized by, The induction culture temperature for the induction expression of step S2 is 16℃ ~ 42℃; the inoculation amount is 0.01% ~ 10%; the inducer is added when the OD600 of the bacterial cells is between 0.1 ~ 0.5; the inducer is IPTG; the final concentration of the IPTG is 0.1 ~ 1mM; the induction time is 2 ~ 10h.

4. The rapid production method according to claim 1, characterized by, The homogenization of step S3 is to add the bacterial suspension into a high-pressure homogenizer, and repeat the homogenization 2 ~ 4 times under the conditions of 15 ~ 20℃, 400 ~ 600bar; incubate the intermediate product after homogenization at 15 ~ 18℃ for 5 ~ 15min; the mass ratio of the bacterial cells to the buffer is 1:5 ~ 8.

5. The rapid production method according to claim 1, characterized by, The amino acid sequence of the recombinant fibronectin is shown in SEQ ID NO.

1.

6. The rapid production method according to claim 1, characterized by, The E. coli of step S2 is BL21DE3 E. coli.

Images