Non-natural amino acid inserted ferritin as well as preparation method and application thereof

Abstract

The invention discloses non-natural amino acid inserted ferritin as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The recombinant ferritin 5F-HFn is efficiently prepared by inserting an unnatural amino acid 4-azido-phenylalanine containing an azido group into the 5th site in a natural amino acid sequence of the human heavy-chain ferritin HFn through a genetic code expansion technology based on termination codon inhibition and combining a double-plasmid transformation and nickel column purification process. A copper-free click reaction is utilized to realize directional coupling with a dibenzocyclooctyne modified DNA fragment or other functional molecules, so that the nano-carrier is endowed with multifunctional characteristics of targeted delivery, biological imaging and the like, and a nano-carrier platform capable of being flexibly configured is constructed.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a non-natural amino acid-intercalated ferritin, its preparation method, and its application. Background Technology

[0002] In the fields of biomedical engineering and nanomaterials, ferritin, as a naturally occurring nanoscale cage-like protein, has become an ideal choice for constructing high-performance nanocarriers due to its unique structural features and biological functions. Human heavy chain ferritin (HFn) is formed by the self-assembly of 24 subunits into a hollow cage-like structure with a diameter of approximately 12 nm. It not only possesses excellent biocompatibility and reversible self-assembly ability, but also specifically binds to transferring receptor 1 (TfR1), which is highly expressed on the surface of tumor cells, exhibiting natural tumor targeting. These properties make it show great application potential in targeted drug delivery, biomedical imaging, and molecular diagnostics. Traditional ferritin functionalization modification techniques mainly rely on surface chemical modification methods, introducing functional molecules through random coupling reactions of cysteine ​​thiol groups or lysine amino groups.

[0003] Due to the inherent characteristics of the natural amino acid sequence, the surface chemical modification of functional molecules through random coupling reactions of cysteine ​​thiol groups or lysine amino groups faces significant challenges in practical applications. Random modification methods cannot precisely control the connection sites of functional molecules, leading to heterogeneous carrier structures, easily disrupting the spatial conformation of ferritin, and consequently affecting its self-assembly properties and binding activity with TfR1, resulting in decreased targeting efficiency and unstable drug loading capacity. Furthermore, complex chemical reaction conditions not only easily introduce toxic byproducts but may also induce protein structural denaturation, severely limiting the safety of ferritin in vivo. In addition, traditional methods struggle to introduce efficient bioorthogonal reaction sites without disrupting the natural structure of ferritin, failing to achieve directional linking of functional molecules and programmable carrier functions, thus hindering the flexible configuration requirements of "one carrier, multiple uses," becoming a key bottleneck restricting the further application of ferritin in the biomedical field. Summary of the Invention

[0004] To address the issues of uncontrollable modification sites, structural heterogeneity, and limited functionality in traditional ferritin, this invention designs and prepares a ferritin with non-natural amino acid insertions. Using a genetic code expansion technique based on stop codon suppression, a non-natural amino acid, 4-azide-phenylalanine hydrochloride (AzF), containing an azide group, is inserted at position 5 of the natural amino acid sequence of human heavy chain ferritin HFn. Combined with dual plasmid transformation and nickel column purification, recombinant ferritin 5F-HFn is efficiently prepared. Copper-free click reaction enables directional coupling with dibenzocyclooctyne (DBCO)-modified DNA fragments or other functional molecules, endowing it with multifunctional properties such as targeted delivery and bioimaging, and constructing a flexibly configurable nanocarrier platform.

[0005] The first aspect of this invention provides a recombinant ferritin 5F-HFn, which is obtained by inserting a non-natural amino acid 4-azido-phenylalanine containing an azide group at position 5 in the natural amino acid sequence of human heavy chain ferritin. Its amino acid sequence is as follows: MTTA X where X It is a non-natural amino acid, AzF.

[0006] A second aspect of the present invention provides a DNA molecule encoding the above-mentioned recombinant ferritin 5F-HFn.

[0007] A third aspect of this invention provides a method for preparing the above-mentioned recombinant ferritin 5F-HFn, comprising the following steps: Step 1: Modify the HFn gene by mutating the codon AGC corresponding to the 5th serine in its natural amino acid sequence to the amber stop codon UAG, and clone it into pET-30a. ® In the vector, an expression plasmid for this mutant ferritin was constructed; Step 2: Construct pEVOL-pAzF carrying an orthogonal aminoacyl-tRNA synthetase gene and a succinate-repressed tRNA gene. ® Helper plasmids; Step 3: Transform the above expression plasmid and helper plasmid together into the host cell to construct and screen positive host bacteria transformed by dual plasmids; Step 4: Identify the positive host bacteria, expand the culture and induce the expression of recombinant protein, collect the expression product, and obtain recombinant ferritin 5F-HFn by disruption, centrifugation and purification.

[0008] Furthermore, in step 3, the host cell is Escherichia coli BL21 (DE3).

[0009] Furthermore, in step 4, the induction of recombinant protein expression involves adding the inducer isopropyl-... β -D-thiogalactoside, L -Arabinose and 4-azido-phenylalanine.

[0010] Furthermore, isopropyl- β -D-thiogalactoside and AzF were at a final concentration of 1 mM. L The final concentration of arabinose was 1.33 mM, and the induction temperature was 37 °C.

[0011] A fourth aspect of the present invention is to provide the use of the above-described recombinant ferritin 5F-HFn in the preparation of diagnostic and / or imaging reagents.

[0012] A fifth aspect of the present invention is to provide the application of the above-mentioned recombinant ferritin 5F-HFn as a drug delivery carrier in the preparation of targeted drugs.

[0013] This invention utilizes a stop codon-based genetic code expansion technique to successfully construct recombinant ferritin 5F-HFn by inserting the non-natural amino acid AzF at position 5 of the natural HFn sequence. This method introduces an azide group with bioorthogonal reactivity without disrupting the natural hollow cage structure of ferritin. This not only preserves its reversible self-assembly properties and TfR1 targeting ability but also provides a precise site for the directional covalent linkage of functional molecules, avoiding the heterogeneity problems caused by random modifications. The accompanying dual plasmid transformation system combined with nickel column affinity purification significantly improves the expression efficiency and purity of the target protein, ensuring high quality and high yield. Furthermore, the copper-free click chemistry reaction between the AzF azide group and the DBCO-modified molecule allows for rapid covalent coupling under physiological conditions without copper ion catalysis, avoiding the potential toxicity of metal ions and making it suitable for precise modification in complex in vivo environments. By specifically binding to functional molecules such as DBCO-modified DNA fragments, programmable configuration for functions such as ferritin targeted delivery and bioimaging is achieved. The diversity of DNA sequences and functional molecules endows the carrier with flexible functional integration capabilities of "one carrier, multiple uses." For example, linking DNA can be used for diagnosis, linking fluorescent groups can be used for imaging, linking drugs can form targeted drugs, and multiple components can be linked simultaneously to achieve integrated diagnosis and treatment. This invention provides an innovative solution for constructing an intelligent nanocarrier platform integrating targeted delivery, controlled drug release, and biodetection, and has significant application value in the fields of targeted drug delivery systems, molecular diagnostic reagents, and biomedical imaging probes. Attached Figure Description

[0014] Figure 1 This is the SDS-PAGE plot of 5F-HFn.

[0015] Figure 2 This is a TEM image of 5F-HFn.

[0016] Figure 3 This is the particle size distribution diagram of 5F-HFn.

[0017] Figure 4 The particle size stability diagram is for 5F-HFn.

[0018] Figure 5 The CD spectrum of 5F-HFn is shown.

[0019] Figure 6 Figure 1 shows the reversible self-assembly behavior of 5F-HFn under different pH conditions.

[0020] Figure 7 1% agarose gel electrophoresis images to detect the connection rate of 5F-HFn and DBCO-30 in Examples 2-6.

[0021] Figure 8 Fluorescence imaging of 5F-HFn-DBCO-FITC uptake by α-mouse hepatic stellate cells at different incubation times. Detailed Implementation

[0022] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0025] This embodiment describes a method for preparing ferritin with non-natural amino acid AzF insertion. The specific steps are as follows: 1. The HFn gene was modified by mutating the codon AGC corresponding to serine at position 5 in its natural amino acid sequence to the amber stop codon UAG, introducing a 6×His tag at the N-terminus, and then cloning it into pET-30a. ® The expression plasmid for this mutant ferritin was constructed using a vector.

[0026] The modified gene nucleotide sequence is as follows: .

[0027] The lowercase letters at the beginning represent the coding DNA sequence corresponding to MHHHHHH, and the lowercase tag in the middle represents the coding DNA sequence corresponding to the stop codon UAG.

[0028] 2. Construct pEVOL-pAzF carrying an orthogonal aminoacyl-tRNA synthetase gene (specifically recognizing AzF) and a succinate-repressive tRNA gene. ® Helper plasmids.

[0029] 3. Thaw the competent BL21(DE3) bacteria on ice, adding 1.5 μL of pET-30a per 100 μL of bacterial culture. ® and 1.5 μL pEVOL-pAzF ®Gently tap to mix, and let stand on ice for 30 min. Then heat shock in a 42 ℃ water bath for 90 s, avoiding shaking during this process. After heat shock, quickly transfer to an ice-water bath and let stand for 2 min. Then add 900 μL of blank LB liquid medium to the centrifuge tube and incubate at 37 ℃ and 150 rpm for 45 min. Finally, plate the transformed E. coli BL21(DE3) on LB agar plates containing kanamycin and chloramphenicol for incubation to screen for positive clones that have successfully undergone double plasmid transformation.

[0030] 4. Select positive clones for activation. Take 2 mL of the activated bacterial culture and add it to 200 mL of LB liquid medium containing 30 μg / mL kanamycin and 34 μg / mL chloramphenicol. Incubate at 37 ℃ and 220 rpm for 16 h.

[0031] 5. Add 1M isopropyl- to the above culture medium. β -D-galactothioglycoside, 20% L - 200 μL each of arabinose and 1M AzF were cultured at 37 ℃ and 220 rpm for 4 h to induce 5F-HFn expression.

[0032] 6. Centrifuge the cultured bacterial suspension at 4 °C and 8000 × g for 3 min to collect all bacterial cells. Resuspend the collected bacterial cells in 10 mL of 1× Binding Buffer (20 mM Tris, 500 mM NaCl, 5 mM imidazole, pH 7.9) containing 1 mM serine protease inhibitor PMSF. Add lysozyme to the resuspension to a final concentration of 1 mg / mL and let stand for 30 min. Then, sonicate the bacterial suspension on ice at 600 W with a cycle of 1 second on / 2 seconds off for 15 min. Afterward, incubate the bacterial suspension in a 60 °C water bath for 20 min to remove heat-labile proteins. Finally, centrifuge the bacterial suspension at 4 °C and 10000 × g for 35 min and collect the supernatant for subsequent experimental procedures.

[0033] 7. Take out a 30 mL resin column and fix it to the iron stand. First, chelate the resin with 20 mL of 80 mM NiSO4 solution, and mix thoroughly with a pipette to ensure that NiSO4 is fully combined with the resin. Then, add 10 mL of the supernatant sample obtained in step 6 to mix the protein with the NiSO4. 2+ Ensure thorough binding. After loading the sample, first equilibrate the nickel column with 10 mL of 1×Binding Buffer solution, then elute with 10 mL of 30 mM imidazole solution to remove impurities, and finally elute with 20 mL of 160 mM imidazole solution to remove the target protein. Collect the eluent.

[0034] 8. Transfer the collected eluent to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa and centrifuge at 8000 × g for 3 min at 4 ℃. After centrifugation, discard the permeate collected in the outer tube and retain the concentrate in the inner tube. Continue ultrafiltration until the liquid volume in the inner tube drops to less than 50% of the initial volume. Then, add Storage Buffer (20 mM Tris-HCl, 0.15 M NaCl, pH 8.0) to the inner tube until it is full, and repeat the ultrafiltration process. When the liquid volume in the inner tube drops to less than 50% of the initial volume after adding the buffer again, the replacement process is complete, and the liquid in the inner tube at this point is the purified 5F-HFn solution.

[0035] The prepared 5F-HFn was characterized below.

[0036] 1. SDS-PAGE characterization of 5F-HFn 5F-HFn was characterized and its purity was analyzed by SDS-PAGE.

[0037] Experimental results are as follows Figure 1 As shown (total protein is the total protein after ultrasonic disruption, lysatesupernatant is the supernatant after water bath, flow-through is the sample ramp-through buffer, wash is 30 mM imidazole elution buffer, 160 mM elution is 160 mM imidazole elution buffer). The theoretical molecular weight of the 5F-HFn subunit is known to be approximately 21 kDa, while... Figure 1 The results showed that the molecular weight of the protein subunits eluted with 160 mM imidazole was also around 21 kDa, and the bands were clear with light impurities, indicating that 5F-HFn was successfully purified by nickel column affinity chromatography in Example 1.

[0038] 2. TEM characterization of 5F-HFn 5F-HFn was subjected to morphological analysis by transmission electron microscopy at a concentration of 0.5 mg / mL.

[0039] Experimental results are as follows Figure 2 As shown, the scale bar is 100 nm. 5F-HFn exhibits a hollow cage-like structure with a particle size of approximately 10 nm.

[0040] 3. Particle size distribution determination of 5F-HFn The particle size distribution of 5F-HFn obtained in Example 1 was determined using a Malvern particle size analyzer.

[0041] Experimental results are as follows Figure 3 As shown in the figure. The average particle size of 5F-HFn was measured to be 12.77 ± 2.123 nm, and its particle size distribution was concentrated, indicating that the sample has good uniformity.

[0042] 4. Determination of particle size stability of 5F-HFn The particle size stability of 5F-HFn was tested using a Malvern particle size analyzer. A sample solution with a concentration of 2 mg / mL was allowed to stand at room temperature, and samples were taken and measured at time points of 0, 12, 24, 60 and 96 h to examine the change in particle size over time.

[0043] Experimental results are as follows Figure 4 As shown, the particle size variation of 5F-HFn was relatively gradual within the observation period of 0–96 h, indicating that it exhibited good stability within 96 h.

[0044] 5. CD spectral determination of 5F-HFn The secondary structure of 5F-HFn was characterized using a circular dichroism spectrometer.

[0045] Experimental results are as follows Figure 5 As shown in the figure, the circular dichroism of 5F-HFn in the wavelength range of 220-280 nm is evident. The significant negative peak at 222 nm indicates that the secondary structure of this protein is mainly α-helical, which is a typical spectral signal of helical structure. This suggests that 5F-HFn retains the typical secondary structure framework of ferritin, and the mutation did not significantly affect its structural stability.

[0046] 6. Evaluation of the reversible self-assembly behavior of 5F-HFn under different pH conditions Purified 5F-HFn at a concentration of 2 mg / mL was divided into 7 aliquots. One aliquot served as the untreated control (group 0), while the remaining 6 aliquots underwent disassembly and self-assembly treatments under different pH conditions. The pH of groups 1, 2, and 3 was adjusted to 2.0, 3.0, and 4.0, respectively, and the pH of groups 4, 5, and 6 was adjusted to 9.0, 10.0, and 11.0, respectively. All groups were incubated at room temperature for 30 min to induce disassembly. Subsequently, the pH of all treated groups was adjusted back to neutral (pH 7.0) to promote self-assembly. Finally, all samples were analyzed by non-denaturing polyacrylamide gel electrophoresis to evaluate the reversible self-assembly behavior of 5F-HFn.

[0047] Experimental results are as follows Figure 6 As shown, groups 2 and 3 treated with acid showed no obvious assembly bands after pH returned to neutral, while group 1 showed partially assembled bands. In the alkaline treatment groups, group 4 showed a band pattern similar to group 0 after pH returned to neutral, indicating completely reversible self-assembly; groups 5 and 6 also showed some degree of assembly bands, but the assembly efficiency or intensity differed from group 4. In summary, the reversible self-assembly of 5F-HFn is pH-dependent, and different degrees of self-assembly can be achieved after treatment under both acidic and alkaline conditions followed by pH return to neutral, with the effect being most significant at pH 9.0. Example 2

[0048] Mix 2 mg / mL 5F-HFn with a 30-base template DNA fragment modified with DBCO (DBCO-30) at a 1:1 molar ratio and incubate overnight at 4 °C to prepare 5F-HFn-DBCO-30. The nucleotide sequence of the DNA is TAATGCTAGTCGATAGTTGAGCTGTCACAG. Example 3

[0049] Same as Example 2, except that the molar ratio of 5F-HFn to DBCO-30 is 1:4. Example 4

[0050] Same as Example 2, except that the molar ratio of 5F-HFn to DBCO-30 is 1:8. Example 5

[0051] Same as Example 2, except that the molar ratio of 5F-HFn to DBCO-30 is 1:16. Example 6

[0052] Same as Example 2, except that the molar ratio of 5F-HFn to DBCO-30 is 1:24. Example 7

[0053] Same as Example 2, except that the molar ratio of 5F-HFn to DBCO-30 is 1:32. Example 8

[0054] Same as Example 2, except that the molar ratio of 5F-HFn to DBCO-30 is 1:48.

[0055] The optimal reaction molar ratio of 5F-HFn to DBCO-30 in Examples 2-8 above was determined by 1% agarose gel electrophoresis, ensuring that the total protein content of each well was consistent during sample loading.

[0056] Experimental results are as follows Figure 7As shown in the figure, agarose gel electrophoresis separates nucleic acid samples by molecular weight. Large molecules experience greater resistance in the gel pores and move more slowly, thus remaining at the top of the gel during electrophoresis. Therefore, the upper band in the figure represents DBCO-30 linked to 5F-HFn, while the lower band represents free DBCO-30. The figure shows that as the molar ratio increases, the brightness of the upper protein-nucleic acid complex band increases, indicating that more protein is linked to DBCO-30. However, when the molar ratio is further increased from 1:24 to 1:48, the brightness of the upper complex band does not change significantly, while the brightness of the lower free DBCO-30 band increases sharply. This phenomenon indicates that under the reaction condition of a molar ratio of 1:24, the effective reaction sites introduced on the surface of the 5F-HFn protein through the non-natural amino acid AzF are approaching saturation. In conclusion, this study determined that the optimal molar ratio for efficient click chemistry reaction between 5F-HFn and DBCO-30 is 1:24. Under these conditions, the protein's reaction sites can be fully utilized, and excessive waste of DBCO-30 can be effectively avoided, providing key experimental parameters for the subsequent precise and efficient loading of functional molecules. Example 9

[0057] Validation of cellular uptake of 5F-HFn-DBCO-FITC DBCO-FITC fluorescent dye was mixed with 5F-HFn and incubated to prepare 5F-HFn-DBCO-FITC using the method in Example 2.

[0058] After mouse hepatic stellate cells (Hsc) were cultured to 80% confluence, they were transferred to 6-well cell culture plates and induced to develop into α-mouse hepatic stellate cells (aHsc) using medium containing 10 ng / mL platelet-derived growth factor-BB (PDGF-BB) for 24 h. This activated Hsc cells were then activated into α-mouse hepatic stellate cells (aHsc), which showed high expression of TfR1 on their surface.

[0059] The product obtained from incubation was centrifuged at 8000 rpm for 3 min using a small ultrafiltration tube (molecular weight cutoff 50 kDa) to remove free DBCO-FITC. Then, it was diluted with serum-free medium to a final concentration of 0.5 mg / mL for 5F-HFn-DBCO-FITC. The activated aHsc was divided into four experimental groups, each receiving the diluted 5F-HFn-DBCO-FITC solution, and incubated at 37 ℃ for 1 h, 2 h, and 4 h, respectively. After incubation, the uptake of 5F-HFn-DBCO-FITC by aHsc was observed and analyzed using a laser confocal microscope.

[0060] Experimental results are as follows Figure 8 As shown, the intensity of FITC fluorescence signal in cells significantly increased with prolonged incubation time, indicating that the uptake of 5F-HFn-DBCO-FITC by aHsc is significantly time-dependent. Furthermore, the fluorescence signal distributions of FITC and TfR1-labeled Alexa647 are highly consistent, suggesting that the targeted uptake of 5F-HFn-DBCO-FITC is driven by TfR1-mediated endocytosis. These results validate the high efficiency and specificity of the 5F-HFn-DBCO complex in targeted cell delivery, providing experimental evidence for its application in tumor-targeted therapy and molecular imaging.

Claims

1. A recombinant ferritin 5F-HFn, characterized in that, The recombinant ferritin is a non-natural amino acid 4-azido-phenylalanine containing an azido group inserted at position 5 in the natural amino acid sequence of human heavy chain ferritin, and the amino acid sequence is: MTTAXTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES, wherein X is a non-natural amino acid AzF.

2. A DNA molecule encoding the recombinant ferritin 5F-HFn of claim 1.

3. The method of producing the recombinant ferritin of claim 1, characterized in that, The method comprises the following steps: Step 1, modifying the HFn gene so that the serine at position 5 in the natural amino acid sequence is mutated from the codon AGC to the amber stop codon UAG, and cloning it into a vector to construct an expression plasmid of the mutant ferritin; Step 2, constructing an auxiliary plasmid carrying the orthogonal aminoacyl tRNA synthetase gene and the amber suppressor tRNA gene; Step 3, co-transforming the above expression plasmid and the auxiliary plasmid into a host cell to construct and screen a positive host bacterium transformed with double plasmids; Step 4, identifying the positive host bacterium, expanding the culture and inducing the expression of the recombinant protein, collecting the expression product, breaking, centrifuging and purifying to obtain the recombinant ferritin 5F-HFn.

4. The production method according to claim 3, characterized by, The host cell in step 3 is Escherichia coli.

5. The preparation method according to claim 3, characterized in that, The induction of expression of the recombinant protein in step 4 is by addition of the inducer isopropyl-β-D-thiogalactopyranoside to the culture medium. β - D-thiogalactoside, L - arabinose and 4-azido-phenylalanine.

6. The production method according to claim 5, characterized by isopropyl- β - Final concentration of D-thiogalactopyranoside, AzF, 1 mM, L - Final concentration of arabinose, 1.33 mM, induction temperature 37 °C.

7. Use of the recombinant ferritin 5F-HFn of claim 1 in the preparation of a diagnostic and / or imaging reagent.

8. Use of the recombinant ferritin 5F-HFn of claim 1 as a drug delivery carrier in the preparation of a targeted drug.

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