Double-compartment protein nanocarriers based on interfacial engineering and their applications

Abstract

The application discloses a double-compartment protein nanocarrier based on interface engineering construction and application thereof, and belongs to the field of food nutrition, biological nanomaterials and protein engineering. The application introduces a zinc binding site in the interface region of a ferritin subunit, realizes specific binding of zinc ions while maintaining the natural iron mineralization function, thereby forming a double-compartment structure coexisting with the interface zinc binding region and the inner cavity iron mineralization region. Compared with the ferritin before modification, the double-compartment ferritin nanocarrier obtained by the application has higher iron and zinc loading capacity, and can be applied to iron and zinc nutrition fortification, functional food development and multi-mineral element delivery system construction.

Description

Technical Field

[0001] This invention relates to dual-compartment protein nanocarriers constructed based on interface engineering and their applications, belonging to the fields of food nutrition, bionanomaterials and protein engineering. Background Technology

[0002] Iron and zinc are two essential trace elements for the human body, playing crucial roles in vital activities such as oxygen transport, immune regulation, cell proliferation, and enzyme catalysis. Iron and zinc deficiency are widespread nutritional problems globally, making the development of safe and effective iron and zinc supplementation systems of great significance.

[0003] Currently, iron and zinc supplements on the market typically use a simple mixing method to achieve compound supplementation. However, iron and zinc ions compete for absorption in the intestines, sharing some transport proteins and absorption pathways, leading to decreased bioavailability. Furthermore, free metal ions are prone to precipitation, oxidation, and binding with food components, further affecting the supplementation's effectiveness.

[0004] Ferritin is a natural nanocage-like protein composed of 24 subunits that self-assemble into a hollow spherical structure. It exhibits good biocompatibility, safety, and natural iron storage capabilities. Natural ferritin primarily stores iron through luminal mineralization; however, its lumen lacks high-affinity binding sites for other minerals such as zinc, copper, and manganese, limiting its application as a multi-mineral delivery carrier. Therefore, developing a novel ferritin delivery system capable of simultaneously loading iron and zinc and achieving spatially separated storage of the two metal ions is of great significance for improving mineral element utilization efficiency. Summary of the Invention

[0005] Technical issues The technical problem to be solved by this invention is that natural ferritin mainly stores iron through luminal mineralization, but its luminal cavity lacks high-affinity binding sites for zinc, which limits its application as a multi-mineral delivery carrier.

[0006] Technical solution This invention provides a dual-compartment ferritin nanocarrier constructed based on interface engineering. By introducing zinc binding sites into the interface region of ferritin subunits, the specific binding of zinc ions is achieved while maintaining the natural iron mineralization function, thereby forming a dual-compartment structure in which the interface zinc binding region and the inner cavity iron mineralization region coexist.

[0007] The dual-compartment ferritin nanocarrier mutates oyster ferritin (GKFN) with the amino acid sequence shown in SEQ ID NO:1, specifically by mutagenesis of E23C, E57C, E58C, C4A, C27A, and C126A, to obtain engineered ferritin GKFN3C. This mutation introduces multiple cysteine ​​residues (cysteine ​​specifically adsorbs metal ions) into the ferritin subunit interface region to construct zinc-binding sites, while retaining the ferritin's natural iron oxidation center and luminal mineralization function; simultaneously, the natural cysteine ​​sites are replaced. The GKFN3C ferritin forms a thiol-rich zinc-binding environment in the subunit interface region, which can be used to bind Zn²⁺. + Simultaneously, it maintains the lumen mineralization function of natural ferritin, achieving Fe²⁺ + Oxidative mineralization deposition occurs within the protein lumen, thus forming an interfacial Zn binding zone and an internal Fe mineralization zone, constituting a dual-compartment mineral element delivery system.

[0008] This invention also provides a method for preparing the dual-compartment ferritin nanocarrier, comprising the following steps: (1) Obtain the gene encoding the ferritin GKFN3C (containing E23C, E57C, E58C, C4A, C27A and C126A mutations), and ligate it into an expression vector to obtain a recombinant plasmid; (2) The recombinant plasmid was transformed into the expression host bacteria to obtain the recombinant bacteria; (3) Culture the recombinant bacteria and induce them to express the ferritin GKFN3C; (4) The ferritin GKFN3C was obtained by purification.

[0009] The present invention provides ferritin that carries both zinc and iron, which is obtained by loading zinc ions and iron ions onto the ferritin GKFN3C.

[0010] The present invention also provides the application of the dual-compartment ferritin nanocarrier and the ferritin that simultaneously carries zinc and iron in nutritional fortifiers, functional foods, dietary supplements, special medical purpose formula foods, and mineral element delivery systems.

[0011] Beneficial effects This invention achieves spatially separated loading of iron and zinc. The cysteine ​​(Cys) side chain contains a thiol group (–SH), which has excellent metal ion coordination ability and can bind with Zn²⁺. + Cu² + Cd² + It forms stable complexes with various metal ions. By introducing cysteine ​​residues on the surface or at specific sites of ferritin through interface engineering to construct zinc-binding regions, while retaining the mineralized regions in the lumen of natural ferritin, it achieves partitioned storage of iron and zinc in the same nanocarrier.

[0012] Compared to the original ferritin, the dual-compartment ferritin nanocarrier obtained in this invention has a higher iron and zinc loading, with the iron loading increased by 41.9% and the zinc loading increased by 23.8%, enabling synergistic delivery of two mineral elements.

[0013] The dual-compartment ferritin nanocarrier obtained by this invention can still maintain the complete nanocage structure and good assembly ability of ferritin, and can maintain the normal iron oxidation and mineralization activity of ferritin after binding with zinc ions.

[0014] The dual-compartment ferritin nanocarrier obtained by this invention has broad application prospects and can be applied to iron and zinc nutritional fortification, functional food development, and the construction of multi-mineral element delivery systems. Attached Figure Description

[0015] Figure 1 Schematic diagram of GKFN3C mutation site design. Figure 2 GKFN and GKFN3C SDS-PAGE and Native-PAGE analysis. Figure 3 Schematic diagram of the GKFN3C dual-compartment iron-zinc load model.

[0016] Figure 4 Zn² + Effect of preloading on the iron oxidation activity of GKFN and GKFN3C. Figure 5 Results of Fe and Zn loading determination in GKFN and GKFN3C.

[0017] Figure 6 GKFN3C molecular docking results. Detailed Implementation

[0018] Example 1: Preparation of engineered ferritin GKFN3C The three-dimensional structure (approximately 12 nm) of oyster ferritin GKFN, with an amino acid sequence as shown in SEQ ID NO:1, was analyzed using the AlphaFold3 structure prediction model. Based on the metal-binding environment of the subunit interface region, Glu23, Glu57, and Glu58 were selected as mutation sites to introduce cysteine ​​residues to construct zinc-binding sites: Glu23Cys, Glu57Cys, and Glu58Cys. Simultaneously, the naturally occurring Cys4, Cys27, and Cys126 were mutated to alanine to avoid non-specific metal binding. The ferritin mutant was named GKFN3C.

[0019] The nucleotide sequence encoding GKFN3C was obtained through gene synthesis and then processed via enzyme cleavage sites. Nde I and XhoI was ligated into the expression vector pET21a. Simultaneously, as a control, the gene encoding unmutated ferritin GKFN was also cleaved at the restriction enzyme site. Nde I and Xho I was ligated into the expression vector pET21a. The resulting recombinant plasmids were transformed into Escherichia coli BL21(DE3) host bacteria to obtain recombinant bacteria for expressing ferritin GKFN3C and GKFN, respectively.

[0020] The recombinant bacteria were cultured at 37℃ until the OD600 reached 0.6–0.8, then IPTG was added to induce expression. The bacterial cells were collected by centrifugation and sonicated. The target protein was purified using a combination of heat treatment and ion exchange chromatography. Protein purity and assembly status were verified by SDS-PAGE and Native-PAGE. Figure 2 As shown, both GKFN and GKFN3C exhibited single protein bands at approximately 17 kDa in SDS-PAGE, indicating successful protein expression and high purity. Native-PAGE results showed that both proteins formed a high-molecular-weight complex of approximately 440 kDa, consistent with the structure of native 24-mer ferritin, demonstrating that the introduction of the cysteine ​​mutation did not impair the protein's self-assembly ability or cage-like structural stability.

[0021] Example 2: Loading iron and zinc onto a dual-compartment ferritin nanocarrier The purified GKFN and GKFN3C proteins were dissolved in 50 mM Tris-HCl (pH 7.5-8.0) buffer to a protein concentration of 2 mg / mL. Zn²⁺ was first added according to a protein to metal ion molar ratio of 50:1. + Vortex for 5 minutes, add the solution 4 times, then use a 10 kDa ultrafiltration tube to remove free Zn²⁺ from the solution. + Subsequently, Fe²⁺ was added according to the protein to metal ion molar ratio (50:1). + Eight additions were made to induce a mineralization reaction, forming an iron core within the ferritin lumen. After dialysis to remove unbound ions, a two-compartment iron-zinc-loaded ferritin was obtained. Simultaneously, wild-type GKFN ferritin was used as a control, and Fe²⁺ was applied under the same conditions. + Mineralization and Zn² + Loading treatment was performed, and the Fe and Zn contents were determined.

[0022] like Figure 3 As shown, engineered ferritin GKFN3C forms Zn²⁺ at the subunit interface region. + The binding zone, while preserving the natural ferritin lumen Fe² + Oxidation and mineralization zone. Zn² + It mainly binds to the cysteine-rich regions formed at the protein shell subunit interfaces, while Fe² +Iron ore cores are formed and stored in the protein lumen after oxidation, thus achieving spatial separation, loading, and storage of iron and zinc in the same protein nanocage.

[0023] Evaluation of Zn² + The effect of preloading on the iron oxidation activity of GKFN and GKFN3C. Figure 4 As shown, Figure 4 A is GKFN, GKFN3C, and its preload of 200 Zn². + Fe² after ionization + Oxidation kinetic curves can be used to evaluate Zn²⁺. + Changes in the catalytic performance of ferritin oxidation centers under pre-loading conditions; Figure 4 B represents the statistical results of the corresponding initial oxidation rate (mean ± standard deviation, n=3). The results indicate that unloaded Zn²⁺… + At that time, the initial oxidation rates of GKFN and GKFN3C were 0.364±0.028 μM / s and 0.347±0.035 μM / s, respectively, indicating that the engineering modification did not significantly affect the oxidation activity of natural iron.

[0024] When preloaded with approximately 200 Zn² + After ionization, the iron oxidation rates of GKFN and GKFN3C decreased to 0.142±0.019 μM / s and 0.270±0.015 μM / s, respectively. GKFN3C retained approximately 77.8% of its iron oxidation activity, while GKFN retained only approximately 39.0%. This indicates that the present invention, by introducing multiple cysteine ​​sites into the subunit interface region to form a Zn-binding region, effectively reduces Zn²⁺ oxidation. + The competitive effect on iron oxidation centers allows the protein to maintain the oxidative mineralization function of natural ferritin while maintaining a high zinc loading capacity.

[0025] The Fe and Zn contents in GKFN and GKFN3C two-compartment ferritin were determined by ICP-MS. All samples were adjusted to the same protein concentration (1 μM) before measurement. Figure 5 As shown, the Zn content in GKFN and GKFN3C is 12.57±0.51 mg / kg and 15.70±0.52 mg / kg, respectively, and the Fe content is 21.97±0.06 mg / kg and 31.27±1.27 mg / kg, respectively.

[0026] Calculations show that GKFN3C has a significantly improved metal loading capacity compared to GKFN, particularly for Zn²⁺. +The loading of Fe and Fe increased by approximately 46 and 140 ions / cage (cage being a single protein), respectively, representing increases of 23.8% and 41.9%. Therefore, the total metal loading increased from 527 ions / cage to 713 ions / cage, an overall improvement of approximately 35.3%.

[0027] Simultaneously, we used AF3 to analyze and simulate docking of individual subunits after mutation. We observed that Cys23, Cys57, and Cys58 all have coordination effects on Zn. Figure 6 ).

[0028] In summary, this invention achieves Zn²⁺ while preserving the natural Fe mineralization zone within the cavity. + By synergistic loading of Fe, an iron-zinc composite nanocarrier with a dual-compartment structure was constructed.

[0029] SEQ ID NO:1 GKFN ESQCRQNYHQESEAGINRQINMELYACYTYQSMAYYFDRDDVALPGFSKFFKNSSDEEREHAEKLMKYQNKRGGRVVLQDIKKPDRDEWGTGLDAMQVALQLEKTVNQSLLDLHKVADSHQDAQMCDFLETHYLEEQVNAIKEISDHITQLKRVGSGLGEYEYDRRLDS SEQ ID NO:2 GKFN3C ESQARQNYHQESEAGINRQINMCLYAAYTYQSMAYYFDRDDVALPGFSKFFKNSSDCCREHAEKLMKYQNKRGGRVVLQDIKKPDRDEWGTGLDAMQVALQLEKTVNQSLLDLHKVADSHQDAQMADFLETHYLEEQVNAIKEISDHITQLKRVGSGLGEYEYDRRLDS Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Ferritin, characterized in that, The oyster ferritin with the amino acid sequence shown in SEQ ID NO:1 was mutated to E23C, E57C, E58C, C4A, C27A and C126A.

2. The ferritin according to claim 1, characterized in that, It has a zinc-bonded region at the subunit interface and an iron mineralization region in the inner cavity.

3. The method for preparing the ferritin according to claim 1, characterized in that, Includes the following steps: (1) Obtain the gene encoding the ferritin and ligate it into an expression vector to obtain a recombinant plasmid; (2) The recombinant plasmid was transformed into the expression host bacteria to obtain the recombinant bacteria; (3) Culturing the recombinant bacteria and inducing them to express the ferritin; (4) Purify to obtain the ferritin.

4. The ferritin of claim 1 loaded with zinc and iron.

5. The ferritin according to claim 4, characterized in that, Zinc content: 15.70 mg / kg; Fe content: 31.27 mg / kg.

6. The use of the ferritin according to claim 1 or 2 in the preparation of nutritional fortifiers, functional foods, dietary supplements, foods for special medical purposes, and mineral delivery systems.

7. The use of the ferritin according to claim 4 or 5 in the preparation of nutritional fortifiers, functional foods, dietary supplements, foods for special medical purposes, and mineral delivery systems.

8. Nutritional fortifiers, functional foods, dietary supplements, foods for special medical purposes, or mineral delivery systems containing the ferritin described in claim 1 or 2.

9. Nutritional fortifiers, functional foods, dietary supplements, foods for special medical purposes, or mineral delivery systems containing the ferritin described in claim 4 or 5.

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