A kind of antifreeze ferritin precisely conjugated with polypeptide and preparation method thereof

Abstract

The present invention discloses an antifreeze ferritin precisely conjugated with a polypeptide and a preparation method thereof, and belongs to the technical field of cryopreservation of biological materials. The ferritin complex prepared by the present invention creatively using gene synthesis can effectively slow down the growth rate of ice crystals, show high-strength ice recrystallization inhibition ability, and the ice crystals formed by the ferritin complex provided by the present invention are uniform in size, rounded on the periphery without sharp edges, and suitable for the preservation of biological materials; the polypeptide is firmly combined with the ferritin core, and no chemical grafting reagent is required, and ferritin is a component of the organism, has no safety hazards, has extremely high biocompatibility, and can be metabolized in the organism; it can be prepared in large quantities by fermentation, with a smaller dosage and lower use cost.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterial cryopreservation, and in particular to an antifreeze ferritin precisely conjugated with a polypeptide and a preparation method thereof. Background Art

[0002] Cryopreservation of biological materials is the foundation of modern biotechnology and biomedicine. For example, the global shortage of transplanted organs has been regarded as a major public health challenge. According to the World Health Organization, only 10% of patients can receive organ transplants. The main reason for this crisis is the short survival time of organs in vitro. In order to avoid damage to organs during the transfer process, the "static cold storage method" is usually used clinically to preserve organs. After the organs are removed from the human body, the preservation fluid is first injected into the organs, and then the organs are placed in a refrigerator. Low temperature is used to slow down the metabolic process of the organs to reduce organ damage. At present, this technology can only preserve donated tissues and organs for a few hours, and the discard rate is very high.

[0003] In order to extend the preservation time of biomaterials and alleviate organ shortages, it is imperative to improve the preservation technology of biomaterials. In addition, the preservation technology of biomaterials is also widely used in other biomedical and biotechnology fields, such as cell therapy, tissue engineering and biobanks.

[0004] It is worth noting that the main challenge in the cryopreservation of biological materials is the uncontrollable formation of ice crystals, which can cause mechanical damage and osmotic stress to organs / tissues / cells, and irreversible damage to intracellular and intercellular structures. Therefore, cryoprotectants must be used to control the growth of ice crystals to minimize damage to cells / tissues during the freezing process.

[0005] In modern medicine, traditional osmotic cryopreservatives (including glycerol and dimethyl sulfoxide) are usually used for cryopreservation, but these cryopreservatives are toxic to biological materials and can cause irreversible damage to the proteins and cell structures in the materials (Liu, Zheng, & Wang, 2022), and can even be fatal at high concentrations.

[0006] In recent years, scientists have extracted natural antifreeze (sugar) proteins from cold-resistant organisms growing in extreme environments (such as polar and plateau environments), which can effectively interact with ice crystals and limit their growth. However, extracting natural antifreeze (sugar) proteins from organisms is a complex and low-yield process, and the unique adsorption mode of antifreeze (sugar) proteins on the prism surface of ice crystals will lead to the formation of needle-shaped ice crystals, which will also cause mechanical damage to cells.

[0007] Through the unremitting efforts of scientific researchers, significant progress has been made in the synthesis of non-toxic and biocompatible bionic antifreeze materials. High molecular polymers (polyproline) that mimic the structure of antifreeze proteins have been reported to have significant ice inhibition activity. By chemically synthesizing different oligopeptides conjugated to the surface of gold nanoparticles of the same size but different morphologies, the growth of ice crystals can be effectively controlled. However, during the synthesis / preparation process of these bionic antifreeze materials, the chemical reagents and heavy metals used—gold nanoparticles—cannot be metabolized in the body by themselves, posing a safety hazard; the synthesis work is time-consuming and costly. Summary of the invention

[0008] The purpose of the present invention is to provide an antifreeze ferritin precisely conjugated with a polypeptide and a preparation method thereof, so as to provide an antifreeze protein that can effectively inhibit the formation of ice crystals, has good biocompatibility, and is free of safety hazards caused by chemical reagents and heavy metals, thereby providing a new material for extending the storage time and quality of biological materials.

[0009] To achieve the above-mentioned object, the present invention provides an antifreeze ferritin with precisely conjugated polypeptides, wherein the polypeptides are polypeptides with potential antifreeze activity; the polypeptides are conjugated to the periphery of the ferritin dodecahedron, with two polypeptides conjugated to each face.

[0010] A method for preparing the antifreeze ferritin precisely conjugated with a polypeptide as described above comprises the following steps:

[0011] S1. preparing a recombinant plasmid containing ferritin and a polypeptide DNA sequence having antifreeze potential;

[0012] S2, transferring the recombinant plasmid into competent E. coli, and then coating it on a solid culture medium with corresponding resistance;

[0013] S3, picking a single bacterial colony and expanding the culture in a liquid culture medium with corresponding resistance;

[0014] S4. Add IPTG to induce protein expression, centrifuge to obtain bacterial sludge, break the cells and centrifuge to obtain the supernatant, precipitate the protein, collect and purify it to obtain antifreeze ferritin precisely conjugated with polypeptide.

[0015] Preferably, the polypeptide with antifreeze potential in step S1 is connected to the N-terminus of the ferritin subunit.

[0016] Preferably, the concentration of IPTG used in step S4 is 100 mg / mL, and the amount added is such that the working concentration of IPTG is 0.5-1 mM.

[0017] Preferably, in step S4, the cells are disrupted by ultrasonic disruption, and the ultrasonic conditions are as follows: power 220-280 W, ultrasonication for 15 min, operation for 3-4 s, and interval of 4-6 s.

[0018] Preferably, in step S4, the protein is precipitated by ammonium sulfate precipitation method, and the addition ratio of ammonium sulfate is 50%.

[0019] Preferably, the purification in step S4 adopts dialysis-DEAE anion exchange column-electrophoresis purification, and the dialysate is 20mM Tris buffer with pH 8.0.

[0020] An application of the antifreeze ferritin precisely conjugated with a polypeptide as described above, wherein the application is in the preservation of biological materials and other work of inhibiting ice crystal growth.

[0021] Ferritin (Ft) is an iron storage protein widely present in organisms, with excellent properties such as low immunogenicity, high biocompatibility and easy degradation. At the same time, as an endogenous protein with a unique cavity and pore structure, Ft has successfully achieved the effective loading of a variety of drug molecules, imaging agents and metal nanoparticles, and is also widely used in multiple biomedical fields such as in vivo delivery, bioimaging, disease diagnosis and treatment.

[0022] Therefore, the present invention provides an antifreeze ferritin precisely conjugated with a polypeptide and a preparation method thereof, and its specific technical effects are as follows:

[0023] (1) The present invention creatively utilizes the method of engineering bacterial expression to connect polypeptide sequence genes to the N-termini of the 24 subunits of ferritin, takes the rigid 12-sided structure of ferritin as the core, and precisely conjugates 24 polypeptide sequences with potential antifreeze activity on its surface. Through the "nail-board" effect, the interaction between the polypeptide and ice crystals at the ice-water interface is enhanced, so that the ferritin complex can be better adsorbed on the surface of ice crystals to achieve efficient antifreeze activity;

[0024] (2) The ferritin complex prepared by the present invention can effectively slow down the growth rate of ice crystals and show a high degree of ice recrystallization inhibition ability. In addition, the ice crystals formed by the ferritin complex provided by the present invention are uniform in size, rounded on the outside and not sharp, and are suitable for the preservation of biological materials.

[0025] (3) The ferritin complex provided by the present invention is prepared by gene synthesis, the polypeptide is firmly bound to the ferritin core, no chemical grafting reagent is required, and ferritin is a component of the organism, has no safety hazards, has extremely high biocompatibility, and can be metabolized in the organism; it can be prepared in large quantities by fermentation;

[0026] (4) The ferritin complex structure provided by the present invention can significantly enhance the ice-inhibiting ability of the polypeptide. The polypeptide that originally required a concentration of 1 mM to exhibit ice-inhibiting ability can achieve the same effect at only 1 μM by conjugating it to ferritin, which is more efficient and has a lower cost of use.

[0027] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0029] Figure 1 is a flow chart of the preparation of the ferritin complex in Example 1 of the present invention;

[0030] Figure 2 is a polyacrylamide gel electrophoresis diagram of the ferritin complex and ferritin in Examples 1 and 2 of the present invention;

[0031] Figure 3 These are transmission electron micrographs and crystal structure diagrams of the electrophoretically pure ferritin complex and ferritin in Experimental Test 1 of the present invention, wherein (A) is a transmission electron micrograph of ferritin, (B) is a transmission electron micrograph of the ferritin complex; (C) is a crystal structure diagram of ferritin; (D) is a crystal structure diagram of the ferritin complex;

[0032] Figure 4 This is a photograph of ice crystals recrystallized in Experimental Test 2 of the present invention;

[0033] Figure 5 This is a photograph of the growth of a single ice crystal in the third experimental test of the present invention. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0035] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0036] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0037] Embodiment 1

[0038] Synthesize an antifreeze ferritin (ferritin complex) with precise peptide conjugation, taking shrimp ferritin (amino acid sequence as shown in SEQ ID NO.1) and the peptide sequence as shown in SEQ ID NO.2 as examples, the preparation process is as follows Figure 1 As shown, the specific steps are as follows:

[0039] S1.1. Send the shrimp ferritin amino acid sequence and polypeptide sequence shown in SEQ ID NO.1 and SEQ ID NO.2 to the company to obtain the shrimp ferritin recombinant plasmid and the recombinant plasmid in which the sequence shown in SEQ ID NO.2 is connected to the N-terminus of the shrimp ferritin subunit (referred to as the shrimp ferritin complex recombinant plasmid).

[0040] S1.2. Spread the received plasmid on a 50μg / mL AMP resistance plate (the resistance of the positive plasmid given by the company), invert and culture at 37℃ until a single colony grows, pick a single clone and culture it in a 37℃ shaker until the OD is 0.6-0.7, then take 2μL of the bacterial solution and send it to the company for DNA sequencing. The sequencing results show that the sequences of the obtained shrimp ferritin recombinant plasmid and shrimp ferritin complex recombinant plasmid are in line with expectations, and the recombinant plasmid is successfully constructed.

[0041] S1.3. Dissolve 10 μL of shrimp ferritin complex recombinant plasmid (concentration of 50 ng / μL) in 100 μL of BL21 competent cells, let stand at 4°C for 30 min, then heat shock the cells at 42°C for 80 s, and then quickly place them in ice for 3 min. After the addition, add 700 μL of sterile LB liquid culture medium, mix well, and incubate in a 37°C incubator for 60 min.

[0042] Take 100 μL of the incubated cells, spread it on LB solid culture medium with 50 μg / mL AMP, and culture it in a 37°C incubator for about 16 hours until a single colony grows. Pick a single colony and dissolve it in 500 mL LB liquid culture medium with 50 μg / mL AMP to expand the culture. When the absorbance of the bacterial solution at 600 nm reaches 0.8, add 360 μL of 100 mg / mL IPTG to induce protein expression. After continuing to culture for 12 hours, centrifuge at 10,000 rpm for 10 minutes to obtain the bacterial sludge.

[0043] S1.4. Resuspend the collected bacterial sludge in Tris buffer (20mM, pH 8.0) and use an ultrasonic disruptor to disrupt the cells. The ultrasonic power is 220-280W, the ultrasonic time is 15min, the working time is 3-4s, and the interval is 4-6s. After the cells are disrupted, centrifuge at 10,000rpm for 10min to obtain the supernatant.

[0044] According to the volume of the supernatant, add ammonium sulfate at a ratio of 50% to precipitate the protein. After the ammonium sulfate is stirred and dissolved, stand at 4°C for 4 hours, centrifuge at 10,000 rpm for 10 minutes to obtain the precipitate. The protein precipitate is redissolved in Tris buffer (20mM, pH8.0) and placed in a dialysis bag. The ammonium sulfate is removed by dialyzing with Tris buffer of the same concentration. The buffer is changed every 6 hours, and the dialysis is repeated three times.

[0045] The dialyzed protein solution was centrifuged at 10,000 rpm for 10 min, the supernatant was carefully collected, filtered with a 4.5 μm pore filter, and passed through a DEAE anion exchange column with Tris buffer to remove impurities. The protein was then eluted with Tris buffer containing a gradient of NaCl. The effluent protein was detected by SDS-PAG E denaturing electrophoresis. The results are shown in Figure 2. Figure 2 The purified ferritin complex was dialyzed with ultrapure water to remove ions, and then freeze-dried for later use.

[0046] The methods of transforming BL21 competent cells, expanding culture, inducing protein expression, protein extraction and purification of shrimp ferritin are exactly the same as those of the shrimp ferritin complex recombinant plasmid. The SDS-PAGE denaturing electrophoresis results of ferritin are shown in Figure 2 shown.

[0047] Depend on Figure 2 It can be seen that the molecular weight of the obtained ferritin is about 19KDa, which is the same as the theoretical size. The molecular weight of the ferritin complex is significantly larger than that of ferritin, which is due to the insertion of a polypeptide sequence at the subunit end of the ferritin complex, proving that the ferritin complex was successfully prepared.

[0048] Experimental Test 1

[0049] The electrophoresis pure protein obtained in Example 1 was observed using a transmission electron microscope as follows:

[0050] The purified ferritin and ferritin complex were adjusted to a concentration of 0.5 mg / mL with Tris buffer, 10 μL was dropped on a clean sealing film, and then the copper mesh covered with a carbon support film was placed face down on the droplet. After 3 minutes, the excess liquid was absorbed with filter paper, and then negatively stained with 2% uranyl acetate for 3 minutes in the same way, and the excess dye was absorbed. After the sample was dried, it was observed with a transmission electron microscope. The results are shown in Figure 2. Figure 3 As shown in (A) and (B), ferritin and the ferritin complex both showed a cage-like structure with a diameter of approximately 12 nm, indicating that the insertion of the polypeptide sequence at the N-terminus would not affect the spatial structure of ferritin.

[0051] The purified protein was concentrated to 10 mg / mL, the protein crystals were cultured, and the crystals were quickly frozen in liquid nitrogen. 25% glycerol was selected as the protective liquid, and the protein crystal diffraction was performed. Combined with molecular dynamics simulation, the results are as follows Figure 3 As shown in (C) and (D), the blue part shows the polypeptide part connected to the N-terminus. It can be seen that the polypeptide sequence is precisely conjugated to the N-terminus of the ferritin subunit.

[0052] Experimental Test 2

[0053] The ice crystal size of the electrophoretically pure ferritin complex and electrophoretically pure ferritin prepared in Example 1 during ice crystal recrystallization was investigated as follows:

[0054] S2.1. The polypeptide described in Example 1 (sequence shown in SEQ ID NO.2) was sent to a company for polypeptide synthesis. The polypeptide was dissolved in 0.9% NaCl to prepare 1 μM and 1 mM free polypeptide solutions, respectively.

[0055] 0.469 mg of the ferritin complex powder purified by electrophoresis in Example 1 was taken and added to 1 mL of 0.9% NaCl solution to prepare a 1 μM ferritin complex solution.

[0056] 0.513 mg of the ferritin powder purified by electrophoresis in Example 1 was taken and added to 1 mL of 0.9% NaCl solution to prepare a 1 μM ferritin solution.

[0057] S2.2, drop 10 μL of the ferritin complex solution prepared in step S2.1 vertically onto a glass slide pre-cooled with liquid nitrogen from a height of 1.6 m, and instantly form a circular ice crystal with a thickness of about 10 μm; then quickly transfer it to a sealed hot and cold stage chamber at -60°C, heat it to -8°C at a rate of 15°C / min, maintain it at -8°C for 30 min, and take a picture of the ice crystals with an optical microscope, as shown in the figure. Figure 4 shown.

[0058] 10 μL of the free peptide solution (1 μM and 1 mM) prepared in step S2.1, 10 μL of 0.9% NaCl solution (as a positive control), and 10 μL of 1 μM ferritin solution were treated in the same manner as above, and the ice crystals were photographed under an optical microscope. Figure 4 As shown, Image J software was used to select Figure 4 The sizes of the ten largest ice crystals in 1 μM ferritin complex solution, 1 μM and 1 mM free polypeptide solutions, 0.9% NaCl solution, and 1 μM ferritin solution were measured and the average values ​​were calculated. The results are shown in Table 2.

[0059] Table 2

[0060] Ice crystal source 10min(μm) 20min(μm) 30min(μm) 0.9% NaCl 123.8±3.3 162.0±7.2 187.5±5.4 1μM free peptide solution 130.2±4.5 147.8±8.7 170.2±9.8 1mM free peptide solution 42.7±6.7 79.8±6.9 86.7±7.5 1 μM ferritin 122.1±2.3 144.1±4.2 165.6±3.2 1 μM ferritin complex 65.3±1.7 83.1±4.1 93.2±2.6

[0061] Depend on Figure 4 As can be seen from Table 2, compared with 0.9% NaCl, 1 μM free polypeptide solution did not show ice recrystallization inhibitory activity, but 1 mM free polypeptide solution showed significant ice inhibition activity, indicating that the ice inhibition activity of the polypeptide is concentration-dependent.

[0062] The electrophoretic pure ferritin complex prepared in Example 1 can show significant ice-inhibiting activity only at a low concentration of 1 μM, and its maximum ice crystal size is 65.3, 83.1, and 93.2 μm at 10, 20, and 30 min, respectively. Compared with the maximum ice crystal size of the negative control group 0.9% NaCl at 10, 20, and 30 min (123.8, 162, and 187.5 μm, respectively), the ice crystal size is reduced by about 50%, while 1 μM ferritin does not show obvious ice-inhibiting activity, which proves that the rigid ferritin as the inner core can effectively enhance the ice binding performance of the polypeptide, thereby showing significant ice-inhibiting activity at low concentrations.

[0063] Experimental Test 3

[0064] The single crystal growth of the ferritin complex solution and the ferritin solution prepared in Example 1 was investigated, and the specific method was as follows:

[0065] 1 μM and 1 mM free polypeptide solutions, 1 μM ferritin solution and 1 μM ferritin complex solution were prepared with 45 wt% sucrose, and 2 μL of each solution was placed on a glass slide coated with silicone oil on all sides, sealed with a coverslip and placed in a closed hot and cold stage chamber, rapidly cooled to -60°C, then gradually cooled until only single ice crystals were seen in the field of view, then slowly heated at a rate of 0.1°C / min, and photographed at 0s, 60s, 120s, and 180s to record the growth of single ice crystals.

[0066] The results are as follows Figure 5 As shown, 1 μM ferritin complex can significantly inhibit the growth rate of single ice crystals.

[0067] Therefore, the ferritin complex creatively prepared by gene synthesis in the present invention can effectively slow down the growth rate of ice crystals and show a high-strength ice recrystallization inhibition ability. The ice crystals formed by the ferritin complex provided by the present invention are uniform in size, rounded on the outside without sharp edges, and suitable for the preservation of biological materials. The polypeptide is firmly combined with the ferritin core, and no chemical grafting reagent is required. Ferritin is a component of the organism, has no safety hazards, has extremely high biocompatibility, and can be metabolized in the organism. It can be prepared in large quantities by fermentation, with a smaller amount and lower cost.

Claims

1. An antifreeze ferritin precisely conjugated with a polypeptide, characterized in that: The polypeptide is a polypeptide with potential antifreeze activity; the polypeptide is conjugated to the periphery of the ferritin dodecahedron, with two polypeptides conjugated to each face; the ferritin is shrimp ferritin; the polypeptide sequence is shown in SEQ ID NO.2; The method for preparing the antifreeze ferritin precisely conjugated with polypeptides comprises the following steps: S1. preparing a recombinant plasmid comprising a DNA sequence of ferritin and a polypeptide having potential antifreeze activity; S2, transferring the recombinant plasmid into competent E. coli, and then coating it on a solid culture medium with corresponding resistance; S3, picking a single bacterial colony and expanding the culture in a liquid culture medium with corresponding resistance; S4, adding IPTG to induce protein expression, centrifuging to obtain bacterial sludge, breaking cells and centrifuging to obtain supernatant, precipitating proteins, collecting and purifying to obtain antifreeze ferritin precisely conjugated with polypeptides; The polypeptide with potential antifreeze activity in step S1 is linked to the N-terminus of the ferritin subunit.

2. The antifreeze ferritin precisely conjugated with a polypeptide according to claim 1, characterized in that: The concentration of IPTG used in step S4 is 100 mg / mL, and the amount added is such that the working concentration of IPTG is 0.5-1 mM.

3. The antifreeze ferritin precisely conjugated with a polypeptide according to claim 1, characterized in that: In step S4, the cells are broken by ultrasonic disruption, and the ultrasonic conditions are as follows: power 220-280 W, ultrasonic 15 min, working 3-4 s, and interval 4-6 s.

4. The antifreeze ferritin precisely conjugated with a polypeptide according to claim 1, characterized in that: In the step S4, the protein is precipitated by ammonium sulfate precipitation method, and the addition ratio of ammonium sulfate is 50%.

5. The antifreeze ferritin precisely conjugated with a polypeptide according to claim 1, characterized in that: The purification in step S4 is carried out by dialysis-DEAE anion exchange column-electrophoresis purification, and the dialysate is a 20 mM Tris buffer solution with a pH of 8.

0.

6. An application of the antifreeze ferritin precisely conjugated with a polypeptide as claimed in any one of claims 1 to 5, characterized in that: The application is application in the preservation of biological materials and other work of inhibiting the growth of ice crystals.

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