Heteromer ferritin capable of embedding insoluble small molecules under mild condition and preparation method of heteromer ferritin

Abstract

The invention provides heteromer ferritin capable of embedding insoluble small molecules under mild conditions and a preparation method of the heteromer ferritin, and relates to the fields of protein gene engineering, food and medicine. According to the invention, human ferritin is taken as a template, and the heteromer ferritin is synthesized and prepared through an escherichia coli double-promoter expression technology. According to the invention, by utilizing the unique property of the heteropolymer ferritin after two different subunits are polymerized, embedding and loading of insoluble small molecules under the mild condition of pH 4.0 are realized, and about 14.7 curcumin molecules or 18.1 adriamycin molecules can be loaded in one heteropolymer ferritin. The heteromer ferritin HLF prepared by the invention has good water solubility, stability and pH response capability, can be depolymerized and recombined under mild conditions, realizes efficient embedding and loading of insoluble micromolecules, greatly improves the photo-thermal stability of the heteromer ferritin HLF, can expand the application of the insoluble micromolecules in the fields of foods and medicines, and has wide application prospects. And a foundation is laid for the storage, in-vivo intake and delivery.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to ferritin for embedding poorly soluble small molecules under mild conditions and a preparation method thereof. Background Art

[0002] Ferritin is an iron storage protein widely found in organisms. It usually appears as a 24-mer in mammals, and each subunit self-assembles into a globulin with a hollow cage structure. Ferritin has good water solubility and stability, and has eight triple axis channels and six quadruple axis channels distributed on its surface, which can realize the exchange of substances and energy between the ferritin cavity and the external environment. Due to its unique and superior properties, ferritin has received great attention in the fields of food and medicine, and is widely used in the embedding, loading, protection and targeted delivery of unstable or poorly soluble substances.

[0003] When using ferritin to load small molecules, there are two main ways to load drugs. One is to directly incubate the small molecules to diffuse into the internal cavity of the protein to achieve loading; the other is to adjust the pH of the solution to 2 or 12, at which time the ferritin subunits will disintegrate and the 24-mer will disintegrate. Then the pH of the solution will be adjusted back to a neutral environment, and the subunits will reassemble into 24-mers. In this process, the small molecules in the solution can be captured and loaded, which is also a classic strategy for ferritin drug loading. However, in this process, due to the harsh pH conditions of the solution, both the ferritin subunits and the small molecules will be damaged to a certain extent, which will lead to the loss of raw materials and guest molecules. Therefore, it is of great practical significance to modify ferritin so that it can disintegrate under mild conditions. Previously, this effect could be achieved by modifying the interface of ferritin or partially deleting its subunits. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a ferritin for embedding curcumin under mild conditions and a preparation method thereof, so as to achieve embedding of curcumin under pH 4 conditions. In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a recombinant protein HLF capable of embedding curcumin under mild conditions and a preparation method thereof. The amino acid sequence of the HLF includes the amino acid sequence shown in SEQ ID NO.1 (including two subunits):

[0006]

[0007] The present invention preferably uses human heteropolymer ferritin (Gene ID: 2512; 2495, PDB Code: 2FHA; 2FFX) as the construction basis. The heteropolymer ferritin is a 24-mer cage protein composed of two subunits, which provides a good structural basis for the embedding of insoluble substances and improving their stability.

[0008] The HLF heteromeric ferritin of the present invention can load about 14.7 curcumin molecules in one ferritin molecule. After being embedded in ferritin, the water solubility of curcumin increases from 11 ng / mL to 5415 ng / mL, and the light and heat stability is also significantly improved.

[0009] The recombinant protein HLF of the present invention can greatly improve the solubility and light and heat stability of curcumin. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The solution characteristics and morphology of recombinant ferritin HLF encapsulated curcumin. Figure A shows the recombinant ferritin HLF solution, curcumin aqueous solution, and the solution of recombinant ferritin HLF encapsulated curcumin (from left to right), and Figure B shows the transmission electron microscopy observation of ferritin HLF encapsulated curcumin;

[0011] Figure 2 After ferritin HLF was embedded with curcumin, the curcumin loading was determined by HPLC;

[0012] Figure 3 Comparison of the photothermal stability of curcumin encapsulated by ferritin and free curcumin in aqueous solution. DETAILED DESCRIPTION

[0013] The present invention provides a ferritin embedded with curcumin under mild conditions and a method for preparing the same. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0014] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention is further described below in conjunction with the embodiments.

[0015] Example 1

[0016] A ferritin encapsulating curcumin under mild conditions and a preparation method thereof, the specific steps are as follows:

[0017] The recombinant protein HLF was expressed and purified using E. coli. Specifically, the plasmid containing the HLF gene (the vector is pRSF-Duet-1) was introduced into BL21 (DE3) E. coli and activated in 5 mL LB medium (37°C, 200r / min, containing 50μg / mL Kana). The activated bacteria were inoculated into 500 mL LB medium for expansion culture (37°C, 200r / min, containing 50μg / mL Kana). When the bacterial cell concentration increased to an OD600 value of about 0.6, IPTG with a final concentration of 1mM was added to induce protein expression, and the bacteria were collected by centrifugation after 8 to 12 hours (6000g, 10min). The collected bacterial precipitate was redissolved in a buffer of 50mM Tris / HCl, pH 8.0, and crushed on an ice bath on an ultrasonic disruptor (ultrasonic time 3s, working interval 4s, ultrasonic duration 15min). The supernatant was centrifuged after sonication (10000g, 10min), the supernatant was heated at 60℃ for 10min to remove some heat-sensitive proteins, and the supernatant was centrifuged again (10000g, 10min). 60% ammonium sulfate was added to the supernatant to precipitate for 6h, the precipitate was collected by centrifugation, and the precipitate was redissolved in 50mM Tris / HCl pH 8.0 buffer. The HLF crude protein was dialyzed in Tris-HCl pH 8.0 solution to remove ammonium sulfate (6h / time). The dialyzed HLF crude protein solution was filtered with a 0.45μm water system membrane for further column chromatography purification. The HLG crude protein solution was further purified using anion exchange column DEAE and gel filtration column S-300. First, the anion column DEAE Sepharose Fast Flow was balanced with buffer A (50mM Tris / HCl, pH 8.0), the HLF crude protein solution was loaded onto the column, and some impurities that were not bound to the filler were washed with buffer A, and then gradient elution (50mM Tris / HCl containing 0-1M NaCl, pH 8.0) was performed at a flow rate of 0.2mL / min, and 2mL was collected in each tube. The collected proteins were detected by SDS-PAGE electrophoresis and the pure protein components were collected. The protein purified by the anion exchange column DEAE was concentrated using a 50kDa ultrafiltration centrifuge tube and purified using a gel filtration column S-300 (50mM Tris / HCl, pH 8.0, 0.15M NaCl) at a flow rate of 0.2mL / min, and 1mL was collected in each tube. Native-PAGE electrophoresis was used to detect the purity and status of each tube of protein, and then the pure protein fractions were collected;

[0018] The purified recombinant protein HLF was concentrated using an ultrafiltration centrifuge tube (50kDa) and then diluted to 2.0μM. 5.0mL 2.0μM HLF protein solution (50mM Tris / HCl, pH 8.0) was placed in a centrifuge tube, the pH value of the solution was adjusted to about 4.0 with HCl, and stirring was maintained for 30min. The prepared curcumin solution (20mM) was slowly added to make the molecular molar ratio HLF: active ingredient 1:100, and the pH of the solution was controlled at 4.0 during the process, and stirring was maintained for 30min. The pH value of the solution was then adjusted to 8.0 with NaOH and stirred for 2h at 4°C. The solution was placed in a dialysis bag and dialyzed three times with 50mM Tris / HCl, pH 8.0 buffer at 4°C, and the dialysis buffer was replaced every 6h to remove free curcumin. The morphology of the recombinant protein HLF after embedding curcumin was analyzed using a transmission electron microscope.

[0019] Example 2

[0020] The recombinant protein HLF improves the solubility of the poorly soluble small molecule doxorubicin after embedding it under mild conditions. The detection wavelength of doxorubicin by ultraviolet-visible spectroscopy is 485nm, and the concentration of doxorubicin molecules is determined by means of ultraviolet-visible spectroscopy. It is calculated that an average of 18.1 doxorubicin molecules can be loaded by one HLF protein.

[0021] Example 3

[0022] The recombinant protein HLF improves the thermal stability of poorly soluble small molecule curcumin after embedding it under mild conditions. The ferritin curcumin inclusion complex obtained in Example 1 was heat treated. The heat treatment conditions were incubation at 60°C in the dark for a total of 45 minutes, and the absorption spectrum changes of the samples were detected using a UV-visible spectrophotometer every 5 minutes. The curcumin mother solution was diluted to an equal concentration with a buffer solution (50mM Tris / HCl, pH8.0) as a control group. The degradation curve of the sample was drawn based on the highest peak of the absorption spectrum.

[0023] Example 4

[0024] The recombinant protein HLF improves the light stability of poorly soluble small molecule curcumin after embedding it under mild conditions. The ferritin curcumin inclusion complex obtained in Example 1 was subjected to light treatment, and the conditions were irradiation at 20 cm from an incandescent lamp at room temperature for 90 minutes, and the absorption spectrum changes of the sample were detected every 10 minutes using a UV-visible spectrophotometer. The curcumin mother solution was diluted to an equal concentration with a buffer solution (50mM Tris / HCl, pH 8.0) as a control group. And the degradation curve of the sample was drawn according to the highest peak of the absorption spectrum.

[0025] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A ferritin for embedding poorly soluble small molecules under mild conditions, characterized in that: The ferritin is human heteromeric ferritin.

2. The ferritin according to claim 1, characterized in that The ferritin cage structure, the poorly soluble small molecule is embedded in the cavity of the ferritin.

3. The ferritin according to claim 2, characterized in that The poorly soluble small molecules include curcumin and doxorubicin.

4. The ferritin according to claim 3, characterized in that The ratio of the ferritin to the embedded curcumin is 1:14.7, and the ratio of the ferritin to the embedded doxorubicin is 1:18.

1.

5. The ferritin according to claim 4, characterized in that The poorly soluble small molecule is embedded by pH-induced ferritin disaggregation and assembly under mild conditions.

6. The ferritin according to claim 5, characterized in that The mild condition is pH 4.0.

Images