CN113444715B - Method for realizing graded embedding and gradual release of binary components through ferritin three-dimensional assembly - Google Patents
Method for realizing graded embedding and gradual release of binary components through ferritin three-dimensional assembly Download PDFInfo
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Abstract
The invention relates to a method for realizing graded embedding and gradual release of binary components through a ferritin three-dimensional assembly. The method comprises the following steps: a) preparing MF2H protein, mutating 158 th threonine and 160 th leucine near the quadriplex channel of ferritin of Penaeus japonicus into histidine to obtain ferritin mutant named as MF2H, separating and purifying to obtain ferritin solution; b) embedding the component 1 into the internal cavity of ferritin by means of pH renaturation; c) adding the component 2 into the protein solution prepared in the step b, adding nickel ions, regulating and controlling ferritin to form a three-dimensional assembly, and embedding the component 2 into an assembled gap; d) the binary component-embedded ferritin three-dimensional assemblies prepared by step c can be gradually released component 2 and component 1 by adding EDTA and adjusting the pH (fig. 1). The ferritin three-dimensional assembly-binary component embedding system prepared by the method can orderly load two molecules into different spaces without mutual interference, can realize separation and contact of the two molecules through construction and disassembly of the assembly and recombinant depolymerization of ferritin, and widens the application range of the ferritin three-dimensional assembly.
Description
Technical Field
The invention relates to a method for realizing graded embedding and gradual release of binary components through a ferritin three-dimensional assembly, and the prepared ferritin three-dimensional assembly-binary component embedding system can realize graded, separated embedding and gradual release of the binary components.
Background
Ferritin molecules widely existing in animals, plants and microorganisms have a hollow structure with an outer diameter of 12nm and an inner diameter of 8 nm. In neutral pH solutions, the native ferritin molecules are usually present as 24-mers, and metal ions and smaller molecules can enter the ferritin lumen via their triaxial and tetraaxial pathways that connect the internal and external environment, while larger molecules need to nick the ferritin cage structure in order to enter the ferritin internal cavity. The ferritin cage is dissociated into subunits under the extremely acidic condition that the pH is less than or equal to 2.0 or the alkaline condition that the pH is more than or equal to 11.0, and recombination is realized when the pH of the solution is adjusted to be neutral again, so that the embedding of macromolecules can be realized smoothly. Thus, the internal cavity of ferritin is an excellent carrier for loading foreign molecules.
The phenomenon of self-assembly of molecules generally refers to the spontaneous transformation of a molecular motif from a disordered state to an ordered state under certain conditions, and is commonly found in various life activities in nature. Self-organization of ferritin moleculesIt is also a focus of scientific research to form three-dimensional assemblies of simple cubic stacks of ferritin when regularly arranged along its three orthogonal quadruple axes. In this three-dimensional assembly, in addition to the internal cavity of ferritin itself, there is another portion of available space, namely the assembly gap between ferritin molecules. The total volume of the unit cell is calculated as
The volume of the internal cavity is about
The volume of the assembly gap is about
Therefore, the assembly gap of the ferritin assembly can be used for loading exogenous molecules, and the volume of the assembly gap is about 3 times of the volume of the cavity in the ferritin, so that more exogenous molecules can be accommodated.
In conclusion, the three-dimensional assembly of ferritin can embed two exogenous molecules in the inner cavity and the assembly gap of the three-dimensional assembly of ferritin simultaneously, so that multi-component regionalized separated embedding is realized, and the embedding of binary components by using the three-dimensional assembly of ferritin is not reported at home and abroad at present.
Disclosure of Invention
Aiming at the technical problems, the invention aims to provide a ferritin three-dimensional assembly-binary component embedding system and a preparation method thereof, which realize the grading, the separated embedding and the gradual release of binary components.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for realizing graded embedding and gradual release of binary components by a ferritin three-dimensional assembly comprises the following steps:
a) preparation of MF2H protein. Mutating 158 th threonine and 160 th leucine near a tetraplex channel of the penaeus japonicus into histidine to obtain a ferritin mutant named as MF2H, and separating and purifying to prepare a 24-polymer ferritin solution with the concentration of 0.5-2 mg/mL;
b) adjusting the pH value of the MF2H protein solution to 3-4, stirring for 10-30 min, adding the component 1 into the solution, stirring for 10-30 min, wherein the ratio of the component 1 to the MF2H is 1: 10-1: 200, adjusting the pH value of the solution to be neutral, and standing at 4-25 ℃ for 10-24 h;
c) adding the component 2 into the protein solution prepared in the step b, wherein the ratio of the component 2 to MF2H is about 1: 10-1: 200, stirring for 5-10 min, uniformly mixing, adding nickel ions, continuously stirring for 5-10 min, and reacting for 12-48 h at 4 ℃ by using a shaking table, so that the component 2 is embedded into an assembly gap, wherein the ratio of MF2H to metallic nickel is 1: 500-1: 1500;
d) and c, adding 1 mM-100 mM EDTA to chelate nickel ions to the binary component embedded ferritin three-dimensional assembly prepared in the step c so as to release the component 2, and then adjusting the pH value of the protein solution to 2-3.5 by using 1M HCl to release the component 1, so that the gradual release of the embedded binary component is realized.
The volume of an assembly gap of the ferritin three-dimensional assembly-binary component embedding system prepared by the preparation method is about 3 times of the volume of an internal cavity of ferritin, so that the embedding proportion of two molecules can be regulated and controlled, the assembly gap can accommodate exogenous molecules with larger volume, and the range of embedded substances is widened.
According to the ferritin three-dimensional assembly-binary component embedding system, the embedding of an internal cavity of ferritin depends on the inherent reversible assembling characteristic of pH regulation of ferritin, and the embedding of assembly gaps is realized by intercepting exogenous molecules into the assembly gaps while the ferritin is self-assembled, so that two molecules can be loaded into different spaces in order without mutual interference.
According to the ferritin three-dimensional assembly-binary component embedding system, the two molecules are isolated from the external environment although the two molecules are in different positions, so that the stability of the ferritin three-dimensional assembly-binary component embedding system can be maintained.
The ferritin three-dimensional assembly-binary component embedding system can realize the contact of two molecules through the disassembly of the assembly and the depolymerization of ferritin, and can regulate the reaction.
The ferritin three-dimensional assembly-binary component embedding system can isolate embedded molecules and simultaneously has a channel connecting two parts of spaces, so that two enzymes in cascade reaction can travel at respective positions to realize cascade catalysis.
Compared with the prior art, the invention has the beneficial effects that:
the ferritin three-dimensional assembly-binary component embedding system prepared by the method can realize grading, separating embedding and gradual release of binary components, has stronger controllability, and widens the application range of the protein three-dimensional assembly.
Drawings
FIG. 1 is a schematic representation of the fractional encapsulation and gradual release of a ferritin three-dimensional assembly.
FIG. 2 is a spectrum diagram showing the change of fluorescence intensity of R6G in the process of hierarchical embedding and gradual release of a ferritin three-dimensional assembly and a diagram showing the change of fluorescence intensity of R6G under ultraviolet light.
(for convenience of publication, the color image of fig. 1 and 2 is changed into black-and-white picture after graying treatment, as can be proved by the drawing at the filing date)
Detailed Description
The invention is further illustrated with reference to the following figures and examples.
The two components are embedded by means of the available storage space of the two parts of the ferritin three-dimensional assembly, namely the internal cavity and the assembly gap of the ferritin, so that the grading, the separated embedding and the gradual release of the binary components are realized.
A method for realizing graded embedding and gradual release of binary components through a ferritin three-dimensional assembly comprises the following steps:
a) preparation of MF2H protein. Mutating threonine 158 and leucine 160 near a tetraploid channel of the ferritin of the penaeus japonicus into histidine to obtain a ferritin mutant named as MF2H, and separating and purifying to prepare a 24-polymer ferritin solution with the concentration of 0.5-2 mg/mL;
b) adjusting the pH value of the MF2H protein solution to 3-4, stirring for 10-30 min, adding the component 1 into the solution, stirring for 10-30 min, wherein the ratio of the component 1 to the MF2H is 1: 10-1: 200, adjusting the pH value of the solution to be neutral, and standing at 4-25 ℃ for 10-24 h;
c) adding the component 2 into the protein solution prepared in the step b, wherein the ratio of the component 2 to MF2H is about 1: 10-1: 200, stirring for 5-10 min, uniformly mixing, adding nickel ions, continuously stirring for 5-10 min, and reacting for 12-48 h at 4 ℃ by using a shaking table, so that the component 2 is embedded into an assembly gap, wherein the ratio of MF2H to metallic nickel is 1: 500-1: 1500;
d) and c, adding 1 mM-100 mM EDTA to chelate nickel ions to the binary component embedded ferritin three-dimensional assembly prepared in the step c so as to release the component 2, and then adjusting the pH value of the protein solution to 2-3.5 by using 1M HCl to release the component 1, so that the gradual release of the embedded binary component is realized.
The volume of the assembly gap of the ferritin three-dimensional assembly-binary component embedding system is about 3 times of the volume of an internal cavity of ferritin, so that the embedding proportion of two molecules can be regulated and controlled, the assembly gap can accommodate exogenous molecules with larger volume, and the range of the embedded substances is widened.
According to the ferritin three-dimensional assembly-binary component embedding system, the embedding of an internal cavity of ferritin depends on the inherent reversible assembling characteristic of pH regulation of ferritin, and the embedding of assembly gaps is realized by intercepting exogenous molecules into the assembly gaps while the ferritin is self-assembled, so that two molecules can be loaded into different spaces in order without mutual interference.
According to the ferritin three-dimensional assembly-binary component embedding system, the two molecules are isolated from the external environment although the two molecules are in different positions, so that the stability of the ferritin three-dimensional assembly-binary component embedding system can be maintained.
The ferritin three-dimensional assembly-binary component embedding system can realize the contact of two molecules through the disassembly of the assembly and the depolymerization of ferritin, and can regulate the reaction.
The ferritin three-dimensional assembly-binary component embedding system can isolate embedded molecules and has a channel connecting two parts of spaces, so that two enzymes which generate cascade reaction can travel at respective positions to realize cascade catalysis.
Examples
The ferritin three-dimensional assembly is used for embedding a fluorescent substance and a quenching agent thereof, and indirectly indicates the implementation of each step of graded embedding and gradual release.
(1) And (3) carrying out graded embedding on the ferritin three-dimensional assembly.
a. And (4) embedding the internal cavity of the ferritin. A certain amount of rhodamine 6G (R6G) is accurately weighed and dissolved in deionized water to prepare a mother solution with the final concentration of 10mM, and the mother solution is stored away from light at 4 ℃ for standby. Approximately 5mL of a 2. mu.M MF2H protein solution (pH 8.0, 50mM Tris-HCl buffer) was placed in a 50mL centrifuge tube with a small internal rotor and stirred slowly. The solution pH was adjusted to 3.0 with 1M HCl, stirred at room temperature for 30min, and the mother liquor R6G was added slowly to give a molar ratio MF2H: R6G of 1: 100, stirring for 20min at room temperature. The solution was then adjusted to pH 8.0 with 1M NaOH and stirred in a chromatographic freezer at 4 ℃ for more than 2 h. Placing the renatured solution in a dialysis bag with the aperture of 12,000-14,000Da, dialyzing with 800mL of Tris-HCl buffer solution (pH 8.0, 50mM) for three times, replacing the dialyzate every 6 hours, and removing free R6G, wherein the step is carried out in a chromatographic refrigerator at 4 ℃ and protected from light; finally, the MF2H solution embedded with R6G is stored at 4 ℃ in the dark for standby.
b. And (3) embedding assembling gaps of the ferritin three-dimensional assembly. A certain amount of Black Hole Quencher-2(BHQ-2) is accurately weighed and dissolved in methanol to prepare mother liquor with the final concentration of 20mM, and the mother liquor is stored at 4 ℃ in a dark place for standby. The MF2H solution in which R6G was embedded was subjected to protein concentration measurement by the Lowry method, and then the protein solution was diluted to a protein concentration of 1. mu.M, and a certain amount of 5M NaCl solution was added to give a final NaCl concentration of 0.3M. Approximately 1mL of a 1. mu.M MF2H @ R6G protein solution (pH 8.0, 50mM Tris-HCl buffer) was placed in a 2mL EP tube, and gently stirred with a small internal rotator. Then 10. mu.L of BHQ-2 mother liquor was added to make the molar ratio of protein to BHQ-2 1: 100, slowly stirred for 10min, then nickel ions are added to make the final concentration 0.5mM, and the mixture is slowly shaken overnight in a chromatographic freezer at 4 ℃ and the procedure is carried out in the dark.
(2) Gradual release of the embedded component.
The obtained assembly embedded with the two components firstly chelates metal ion nickel through EDTA to enable three-dimensional assembly to be disassembled, BHQ-2 embedded in an assembly gap is released, and then the pH value of the solution is adjusted to 3.5 to enable the ferritin nanocage to be disassembled, so that fluorescent molecules R6G embedded in the inner cavity of ferritin are released.
(3) The fluorescence of R6G was monitored in each of the above steps, and the progress of each step was indicated by a change in the fluorescence intensity of R6G. The specific results are shown in FIG. 2. As can be seen from the figure, in the protein solution in which R6G is embedded
Adding BHQ-2
Then, the fluorescence intensity of R6G was reduced, and nickel ions were added to embed BHQ-2 in the assembly gap
After that, the fluorescence intensity further decreases. When assembled and disassembled
Ferritin dissociation (
pH 3.5), the fluorescence of R6G was almost completely quenched.
Claims (6)
1. A method for realizing graded embedding and gradual release of binary components through a ferritin three-dimensional assembly is characterized in that: the method comprises the following steps:
a) preparation of MF2H protein: mutating threonine 158 and leucine 160 near a tetraploid channel of the ferritin of the penaeus japonicus into histidine to obtain a ferritin mutant named as MF2H, and separating and purifying to prepare a 24-polymer ferritin solution with the concentration of 0.5-2 mg/mL;
b) adjusting the pH value of the MF2H protein solution to 3-4, stirring for 10-30 min, adding the component 1 into the solution, stirring for 10-30 min, wherein the ratio of the component 1 to the MF2H is about 1: 10-1: 200, adjusting the pH value of the solution to be neutral, and standing at 4-25 ℃ for 10-24 h;
c) adding the component 2 into the protein solution prepared in the step b, wherein the ratio of the component 2 to MF2H is about 1: 10-1: 200, stirring for 5-10 min, uniformly mixing, adding nickel ions, continuously stirring for 5-10 min, and reacting for 12-48 h at 4 ℃ in a shaking table, so that the component 2 is embedded into an assembly gap, wherein the ratio of MF2H to metallic nickel is 1: 500-1: 1500;
d) c, adding 1 mM-100 mM EDTA to chelate nickel ions to the binary component embedded ferritin three-dimensional assembly prepared in the step c so as to release the component 2, and then adjusting the pH value of the protein solution to 2-3.5 by using 1M HCl so as to release the component 1, thereby realizing gradual release of the embedded binary component;
wherein component 1 and component 2 are capable of reacting and are selected from the group consisting of enzymes and substrates, fluorescent substances and quenchers, enzymes that undergo a cascade reaction.
2. A ferritin three-dimensional assembly-binary component encapsulation system prepared according to the method of claim 1, wherein: the volume of the assembly gap is about 3 times of the volume of the internal cavity of the ferritin, so that the embedding proportion of two molecules can be regulated and controlled, the assembly gap can accommodate exogenous molecules with larger volume, and the range of the embedded substances is widened; the length, width and height of the embedding component 1 are not more than 8nm, and the length, width and height of the component 2 are not more than 12 nm.
3. The ferritin three-dimensional assembly-binary component embedding system prepared according to the method of claim 1, wherein: the embedding of the internal cavity of the ferritin depends on the inherent reversible assembly characteristic of pH regulation of the ferritin, and the embedding of the assembly gaps is to intercept exogenous molecules into the assembly gaps while the ferritin is self-assembled, so that the two molecules can be loaded into different spaces in order without mutual interference.
4. The ferritin three-dimensional assembly-binary component embedding system prepared according to the method of claim 1, wherein: the two molecules are in different positions, but are isolated from the external environment, so that the stability of the two molecules can be maintained.
5. The ferritin three-dimensional assembly-binary component embedding system prepared according to the method of claim 1, wherein: the contact of two molecules can be realized by disassembling the assembly and depolymerizing ferritin, and the reaction can be regulated and controlled.
6. The ferritin three-dimensional assembly-binary component embedding system prepared according to the method of claim 1, wherein: the embedded molecules can be isolated, and the channel connecting the two parts of spaces is provided, so that the two enzymes which have cascade reaction can move upwards at respective positions to realize cascade catalysis.
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