A controllable protein recruitment system based on artificial protein cages and a construction method thereof

Abstract

The present invention discloses a controllable protein recruitment system based on artificial protein cages and a construction method thereof, belonging to the technical fields of genetic engineering and bioengineering. In the present invention, Mi3 is used as a scaffold protein, and an FKBP short peptide is connected to the scaffold protein by gene fusion, and the outer surface of Mi3 is modified to obtain a Mi3 that responds to rapamycin stimulation chem ; meanwhile, by utilizing the property that the small molecule rapamycin mediates the non-covalent heterodimerization of FK506-binding protein (FKBP) and FRB, in the case of adding rapamycin, the recruitment of cargo proteins with FRB tags on Mi3 chem is realized, and good recruitment effects are achieved in both extracellular and intracellularly constructed protein cages. The present invention realizes a controllable recruitment process for target proteins, providing new ideas and new ways for efficient production.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and bioengineering, and particularly relates to a controllable protein recruitment system based on artificial protein cages constructed intracellularly and extracellularly and a construction method thereof. Background Art

[0002] Protein cages are self-assembled, monodisperse three-dimensional structures. Most protein cages are spherical, and there are also other shapes such as rod-shaped, ring-shaped or more complex geometric shapes. In the long process of evolution, nature has formed many proteins that can self-assemble into cage-like structures, including heat shock proteins, chaperone proteins, ferritin, and viroid-like particles. They are self-assembled from multiple copies of single or multiple protein subunits into complex supramolecular structures. They have distinct inner and outer surfaces and the interfaces between subunits. The internal space can form a natural physical barrier with the external environment to protect the cargo, the outer surface can interact with cells for infection and immune defense, and the interfaces between subunits play a key role in the assembly of protein cages and affect the stability of the cage-like structure. The precise structural and functional characteristics of its viroid capsid have broad application prospects in the biomedical field, especially in gene delivery and biomimetic viruses, and have received in-depth attention from the academic community in recent years. Inspired by this, many highly symmetric and complex protein cages have been designed from scratch based on natural protein cages, such as I301, TRAP-cage, I52-32, etc. They have good biocompatibility, stability and modifiability.

[0003] Protein cages have been used to encapsulate enzymes for cascade reactions to enhance the overall metabolic flux of synthetic pathways and have also been used as vaccine and drug carriers. For example, Wei Kang et al. (W. Kang, X. Ma, D. Kakarla, H. Zhang, Y. Fang, B. Chen, K. Zhu, D. Zheng, Z. Wu, B. Li, C. Xue, Angew. Chem. Int. Ed. 2022, 61, e202214001; Angew. Chem. 2022, 134, e202214001.) used protein cages as scaffolds to recruit sequential enzymes in the lycopene pathway to the outer surface of the protein cages through SpyTag and SpyCatcher, thereby increasing the lycopene yield by eightfold. However, most current protein cages recruit proteins in an uncontrollable manner. In addition, protein cages have been designed to regulate the recruitment of cargo proteins through pH (Brasch M, Putri RM, de Ruiter MV, Luque D, Koay MS, Castón JR, Cornelissen JJ. Assembling Enzymatic Cascade Pathways inside Virus-Based Nanocages Using Dual-Tasking Nucleic Acid Tags. J Am Chem Soc. 2017 Feb 1; 139(4):1512 - 1519.); or the recruitment of cargo is achieved by the electrostatic interaction between the cargo protein and the protein cage ( B, Pianowski Z, Hilvert D. Efficient in vitro encapsulation of protein cargo by an engineered protein container. J Am Chem Soc. 2012 Jan 18; 134(2):909 - 11.). However, their scope of application is limited. Therefore, there is an urgent need to research and develop a protein cage system that can respond to external stimuli for controllable recruitment and its construction method. Summary of the Invention

[0004] In view of the fact that most existing protein recruitment methods are uncontrollable and the few controllable methods have the disadvantage of poor recruitment stability. The purpose of the present invention is to provide a controllable, stable, and relatively fast protein recruitment system for artificial protein cages and its construction method.

[0005] The present invention provides the following technical solutions:

[0006] The first object of the present invention is to provide a method for constructing an extracellular controllable protein recruitment system based on artificial protein cages, which mainly includes the following steps:

[0007] (1) Connect the gene fragment of the protein cage Mi3 subunit with the gene fragment of FK506 binding protein (FKBP) through a flexible linker sequence to obtain a recombinant gene fragment capable of expressing FKBP-Mi3, insert it into a plasmid vector, and construct a plasmid capable of expressing the protein cage Mi3 in a host cell, transform it into the host cell, and after culturing and inducing expression, isolate and purify to obtain the protein cage Mi3 chem ; chem ;

[0008] (2) Connect the gene fragment of the FRB domain with the gene fragment of the target protein through a flexible linker sequence to obtain a recombinant gene fragment capable of expressing the target protein molecule with the FRB domain, insert it into a plasmid vector, construct a plasmid capable of expressing the target protein molecule with the FRB domain in a host cell, transform it into the host cell, and after culturing and inducing expression, isolate and purify to obtain the target protein molecule with the FRB domain;

[0009] (3) Mix the protein cage Mi3 obtained in step (1) chem with the target protein molecule with the FRB domain obtained in step (2), then add rapamycin for controllable recruitment, and isolate and obtain the controllable protein recruitment system by size exclusion chromatography (SEC).

[0010] Based on the above technical solution, further, the sequence of the FK506 binding protein (FKBP) in step (1) is located at the N-terminus of the protein cage Mi3 subunit sequence, and the amino acid sequence of the protein cage Mi3 subunit is the 133-337th positions in SEQ ID NO.1, and the amino acid sequence of the FK506 binding protein (FKBP) is the 14-120th positions in SEQ ID NO.1.

[0011] Based on the above technical solution, further, the amino acid sequence encoded by the flexible linker sequence in step (1) is (GGS) n or (GGGGS) n , n = 1-10, preferably (GGS)4, and the amino acid sequence is the 121-132nd positions in SEQ ID NO:1.

[0012] Based on the above technical solution, further, the host cells in step (1) include Escherichia coli, yeast, and animal cells, preferably Escherichia coli BL21(DE3).

[0013] Based on the above technical solution, further, the plasmid described in step (1) is a vector with a T7 promoter, which can highly express the target protein in host cells after induction with isopropyl β-D-thiogalactoside (IPTG), and pET28a is preferably used.

[0014] Based on the above technical solution, further, the protein cage Mi3 in step (1) chem has its gene inserted after the T7 promoter of the plasmid vector.

[0015] Based on the above technical solution, further, when the host cell is Escherichia coli in step (1), antibiotics corresponding to the used plasmid are added to the LB medium, and 50 mg / L kanamycin sulfate is preferably used.

[0016] Based on the above technical solution, further, the OD of the host cell described in step (1) 600 reaches 0.4 - 0.8, and 0.1 - 1 mM IPTG is added to induce protein expression, and the optimal concentration is 0.3 mM.

[0017] Based on the above technical solution, the protein cage Mi3 in step (1) chem carries a 6×His-tag label and is purified using a Ni Sepharose High Performance chromatography column.

[0018] Based on the above technical solution, further, the target proteins in step (2) include fluorescent proteins mCherry, CFP, and YFP; the amino acid sequence of mCherry is shown as positions 14 - 248 in SEQ ID NO.2; the nucleotide sequence of CFP is shown as positions 14 - 252 in SEQ ID NO.3; the nucleotide sequence of YFP is shown as positions 127 - 363 in SEQ ID NO.4;

[0019] Based on the above technical solution, further, the FRB domain in step (2) can specifically bind to the FK506 binding protein (FKBP), and the amino acid sequence of FRB is positions 261 - 355 in SEQ ID NO.2;

[0020] Based on the above technical solution, further, the amino acid sequence encoded by the flexible linker sequence in step (2) is (GGS) n or (GGGGS) n , n = 1 - 10, and (GGS)4 is preferably used, and the amino acid sequence is positions 249 - 260 in SEQ ID NO:2;

[0021] Based on the above technical solution, the host cells described in step (2) include Escherichia coli, yeast, and animal cells, preferably Escherichia coli BL21(DE3).

[0022] Based on the above technical solution, the plasmid described in step (2) is a vector with a T7 promoter, and after induction with isopropyl β-D-thiogalactoside (IPTG), it can highly express the target protein in host cells, preferably pCDF-Duet-1.

[0023] Based on the above technical solution, further, the gene of the target protein molecule described in step (2) is inserted after the T7 promoter of the plasmid vector.

[0024] Based on the above technical solution, further, when the host cell is Escherichia coli in step (2), the LB medium is added with the antibiotic corresponding to the used plasmid, preferably 100 mg / L streptomycin sulfate.

[0025] Based on the above technical solution, further, the OD 600 of the host cell described in step (2) reaches 0.4 - 0.8, and 0.1 - 1 mM IPTG is added to induce protein expression, and the optimal concentration is 0.3 mM.

[0026] Based on the above technical solution, further, the target protein molecule with an FRB domain described in step (2) carries a 6×His tag, and Ni Sepharose High Performance chromatography column is used for purification.

[0027] Based on the above technical solution, further, the concentration of rapamycin described in step (3) is 2 - 20 μM.

[0028] Based on the above technical solution, further, in step (3), the molar ratio of the protein cage Mi3 chem to the target protein molecule with an FRB domain is 1:1 - 1:3. Preferably, the molar ratio of the protein cage Mi3 chem to the target protein molecule with an FRB domain is 1:3.

[0029] The present invention also provides a controllable protein recruitment system obtained by the above construction method.

[0030] The present invention uses the protein cage Mi3 as a scaffold, and utilizes the characteristic that FKBP and FRB spontaneously form a non-covalent bond in the presence of rapamycin. By genetically fusing and expressing FKBP, its surface can be modified, enabling Mi3 chem protein cage to recruit cargo proteins with FRB, and further obtaining a controllable protein recruitment system.

[0031] The second object of the present invention is to provide a method for constructing a controllable protein recruitment system in host cells based on artificial protein cages, which mainly includes the following steps:

[0032] (1) Select plasmid vectors that can coexist in host cells, and insert the FKBP-Mi3 recombinant gene fragment in which the gene fragment of FK506 binding protein (FKBP) and the gene fragment of the Mi3 subunit of the protein cage are connected by a flexible linker sequence, and the recombinant gene fragment of the protein molecule with the FRB domain in which the gene fragment of the FRB domain and the gene fragment of the target protein are connected by a flexible linker sequence;

[0033] (2) Transform the recombinant plasmids obtained in step (1) into the same host cell together to obtain a host cell capable of simultaneously expressing the protein cage Mi3 chem and the protein molecule with the FRB domain;

[0034] (3) Culture the host cell obtained in step (2) and induce the expression of the protein cage Mi3 chem and the protein molecule with the FRB domain, and then add rapamycin for controllable recruitment to construct a controllable protein recruitment system in the host cell.

[0035] Based on the above technical solution, further, the FKBP-Mi3 recombinant gene fragment in step (1) is inserted between the NcoI and XhoI sites of the vector pET28a, and the recombinant gene fragment of the protein molecule with the FRB domain is inserted between the NcoI and BamHI sites of the pCDF-Duet-1 plasmid.

[0036] Based on the above technical solution, further, the coexisting plasmid vectors in step (1) have different replication origins and different resistance genes.

[0037] Based on the above technical solution, further, the host cells in step (2) include Escherichia coli, yeast cells and animal cells, preferably Escherichia coli BL21(DE3).

[0038] Based on the above technical solution, further, the LB medium in step (3) is added with the antibiotic corresponding to the plasmid used.

[0039] Based on the above technical solution, further, when the host cell is Escherichia coli in step (2), the LB medium is added with the antibiotic corresponding to the plasmid used, preferably 50 mg / L kanamycin sulfate and 100 mg / L spectinomycin sulfate.

[0040] Based on the above technical solution, further, the OD of the Escherichia coli engineering strain in step (3) 600When it reaches 0.4 - 1.0, add 0.1 - 1 mM IPTG to induce protein expression, and the preferred concentration is 0.3 mM.

[0041] The beneficial effects of the present invention compared with the prior art are as follows:

[0042] 1. The present invention develops an extracellular controllable protein recruitment system based on artificial protein cages. This system can achieve the effect of controllable recruitment of model proteins extracellularly, with a stable structure and good catalytic effect.

[0043] 2. The present invention develops a method for an intracellular controllable protein recruitment system based on artificial protein cages. The method can achieve the effect of controllable recruitment of model proteins intracellularly, and the method can be widely applied.

[0044] 3. The chemically sensitive protein cage Mi3 disclosed in the present invention chem , after adding rapamycin, can specifically recruit the target protein with an FRB domain. Therefore, the recruitment of the target protein by the protein cage can be rapidly and precisely regulated by adding the small molecule rapamycin.

[0045] 4. The protein cage subunit of the present invention is a virus-like particle (VLP), which can automatically assemble into a protein cage. Therefore, it is suitable as a protein recruitment scaffold to construct a controllable protein recruitment system, and has the following advantages: (1) The assembly process of Mi3 has good robustness and is easy to chemically modify; (2) Mi3 is self-assembled by 60 subunits and has a high enzyme loading capacity; (3) Mi3 has a high-resolution protein structure, which can achieve the precise assembly of enzyme molecules at the atomic level. (4) The recruitment of the target protein is controllable. Brief Description of the Drawings

[0046] To more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments will be briefly introduced below.

[0047] Figure 1 It is a schematic diagram of the controllable self-assembly of the controllable protein recruitment system based on artificial protein cages of the present invention.

[0048] Figure 2 It is the SEC and SDS-PAGE diagrams of the controllable protein recruitment system based on artificial protein cages in Example 1. (A) is the detection of the assembly of Mi3 chem and FRB-mCherry by gel chromatography; (B) is the SDS-PAGE protein electrophoresis test of the in vitro assembly results. In the experimental group, peak 1 is the in vitro assembly of Mi3 chem and FRB-mCherry at a molar ratio of the target protein of 1:3; peak 2 is the excess cargo protein FRB-mCherry, M is the Marker, and in the control group, peak 1: Mi3 chem; Peak 2: FRB-mCherry.

[0049] Figure 3 is Mi3 chem Transmission electron micrograph of the protein cage and the controllable protein recruitment system, with a scale bar of 100 nm.

[0050] Figure 4 is the protein cage Mi3 chem Graphs of dynamic light scattering analysis before and after assembly with the protein molecule FRB-mCherry.

[0051] Figure 5 Graph showing the assembly results of Mi3chem and FRB-CFP in cells observed by laser scanning confocal microscopy (scale bar: 4 μm).

[0052] Figure 6 Graph of CFP fluorescence lifetime detected by fluorescence lifetime imaging experiment in the experimental group and the control group. Detailed implementation manners

[0053] To facilitate the understanding of the present invention, the present invention will be described in detail below in conjunction with embodiments. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive, but the implementation manners of the present invention are not limited thereto.

[0054] The following further elaborates on the present invention through specific embodiments, but does not limit the protection scope of the present invention.

[0055] Example 1: Protein cage Mi3 linked with a FK506-binding protein (FKBP) domain chem Construction of engineering strains with protein molecules mCherry-FRB, CFP-FRB, and FRB-YFP

[0056] When overexpressing proteins, BL21(DE3) engineering bacteria are selected. The chromosome of this strain integrates the phage DE3 region, which contains the T7 RNA polymerase gene and its promoter lacUV5, and can express T7 RNAP; moreover, this strain lacks Lon protease and OmpT protease, which can reduce the degradation of recombinant proteins. In addition, the lac operon structure is used in combination with the T7 promoter, enabling the expression vector to only highly express the target protein when induced by IPTG or lactose, while maintaining a low background expression level when not induced, avoiding the influence on the stability of cells and plasmids due to overexpression of the target gene. The protein cage Mi3 linked with the FKBP domain chemThe amino acid sequence of the subunit is shown in SEQ ID NO:1, the amino acid sequence of the protein molecule mCherry-FRB is shown in SEQ ID NO:2, the amino acid sequence of the protein molecule CFP-FRB is shown in SEQ ID NO:3, and the amino acid sequence of the protein molecule YFP-FRB is shown in SEQ ID NO:4.

[0057] The Mi3chem gene of the protein cage subunit linked with the FKBP domain was inserted between the NcoI and XhoI sites of the plasmid pET28a by homologous recombination. The genes of the protein molecules mCherry-FRB and CFP-FRB were respectively inserted between the NcoI and BamHI sites of the plasmid pCDF-Duet1 by homologous recombination. At the same time, the YFP-FRB gene was inserted between the EcoRV and XhoI sites of the plasmid pCDF-Duet1-FRB-CFP by homologous recombination. Then the obtained plasmids were respectively introduced into the Escherichia coli cells BL21(DE3), so as to obtain engineering strains capable of expressing the protein cage Mi3chem and the protein molecules FRB-mCherry, FRB-CFP / YFP-FRB respectively.

[0058] Example 2: In vitro construction of a controllable protein recruitment system based on a protein cage, including the following steps:

[0059] (1) Inoculate the engineering strain of the protein cage subunit Mi3chem linked with the FKBP domain in Example 1 into 1 L of LB medium containing 50 mg / L kanamycin sulfate; inoculate the engineering strain of the protein molecule FRB-mCherry into 1 L of LB medium containing 100 mg / L spectinomycin sulfate, and culture both at 37 °C and 220 rpm. When the OD 600 of the Escherichia coli fermentation broth reaches 0.6, add IPTG (final concentration is 0.3 mM for both) to induce the expression of related proteins, and culture overnight at 24 °C. Centrifuge the overnight-induced Escherichia coli fermentation broth at 12,000 g, collect the cells, and discard the supernatant;

[0060] (2) Resuspend the bacterial cells with 50 ml of binding buffer (20 mM Tris, 150 mM NaCl, 20 mM imidazole, pH = 7.8), and disrupt the cells using a high-pressure homogenizer for 10 min. Resuspend the disrupted cell suspension and centrifuge it at 12,000 g for 20 min. Collect the supernatant and filter it through a 0.22-μm filter membrane. Bind the target protein in the supernatant using a nickel column, and linearly elute the target protein with elution buffer (20 mM Tris, 150 mM NaCl, 500 mM imidazole, pH = 7.8). Finally, further separate the target protein by size-exclusion chromatography (SEC) to obtain the purified protein cage Mi3chem and the protein molecule mCherry-FRB respectively;

[0061] (3) In a PBS buffer environment (20 mM Tris, 150 mM NaCl, pH = 7.4), mix Mi3 chem and FRB-mCherry extracellularly in a centrifuge tube at a molar ratio of target protein 1:3, add 2 μM rapamycin, and pipette to mix evenly. Let it stand at 25 °C for 12 h; rely on the spontaneous binding of FKBP / FRB to form a complex in the presence of rapamycin, thereby constructing the controllable protein assembly system based on protein cages extracellularly (as Figure 2 shown).

[0062] Subsequently, perform fusion protein characterization: Under the condition of adding 2 μM rapamycin to the above-mentioned protein cage Mi3chem and protein molecule mCherry-FRB, separate the assembled complex using gel chromatography technology (the results are as Figure 2 shown in A). Peak 1 indicates that Mi3 chem has successfully self-assembled, and peak 2 is the excess protein molecule mCherry-FRB; Subsequently, perform SDS-PAGE on the purified Mi3 chem protein cage (subunit 36.7 kDa) and FRB-mCherry (41.3 kDa) (as Figure 2 shown in B), in which both the Mi3 chem protein cage and FRB-mCherry show a single band, and the assembled complex has two bands because FKBP and FRB are non-covalently bound, and high temperature will break the non-covalent bond, so there are two bands in the lane;

[0063] Subsequently, characterize the Mi3 Figure 3 protein cage and the assembled complex by transmission electron microscopy (TEM) (as chem shown). It can be seen that Mi3 chemThe protein cage presents a uniform spherical structure with a uniform particle size. After coupling with the mCherry-FRB protein molecule, the contour becomes more blurred and the particle size increases. Subsequently, the particle size of Mi3 was measured using dynamic light scattering technology. chem The particle size of Mi3 chem was about 30.1 nm, and the particle size of the assembled complex was about 36.4 nm, with a very good degree of uniformity, presenting a stable cage structure, and the particle size increased compared with that before assembly, which was consistent with the theory (as Figure 4 shown). The above results prove that a controllable protein recruitment system based on artificial protein cages has been successfully constructed.

[0064] Example 3: Apply this assembly method to Escherichia coli cells to explore its assembly effect in cells, including the following steps:

[0065] Insert the CFP gene with FRB (FRB-CFP) and the YFP gene with FRB (FRB-YFP) into the plasmid pCDFDuet-1 between the NcoI and BamHI sites and between the EcoRV and XhoI sites respectively to construct a plasmid that can express FRB-CFP / YFP-FRB. Insert the gene of the protein cage Mi3 chem into the plasmid pET28a between the NcoI and XhoI sites to construct a plasmid that can express the protein cage Mi3 chem . Introduce the two constructed plasmids into Escherichia coli BL21(DE3) cells to obtain an engineered strain Strain 1 that can express the corresponding proteins under IPTG induction, thereby constructing the protein assembly system in the cells. Culture the strain in 20 mL of LB medium containing 50 mg / L kanamycin sulfate and 100 mg / L spectinomycin sulfate until the OD 600 reaches 0.6, add 0.3 mM IPTG and then culture at 37 °C for 3 h to induce protein expression.

[0066] For 20 mL of Escherichia coli that has expressed the corresponding proteins, add 10 mL of the bacterial solution to rapamycin with a final concentration of 2 μM as the experimental group, and add 10 mL of the bacterial solution to DMSO with a final concentration of 2 μM as the control group. Place them in a shaker at 37 °C and culture for 5 min. Then take 1.5 μL of each bacterial solution to make samples, and use a laser scanning confocal microscope to observe the assembly effect in the cells. If Mi3 chem successfully recruits the cargo protein, the fluorescent protein will aggregate at both ends of the cell. If Mi3 chem does not recruit the cargo protein, the fluorescent protein will be diffused in the cytoplasm.

[0067] Figure 5 Figure for observing the intracellular Mi3 in the experimental group by laser scanning confocal microscope chemThe fluorescence localization of the protein molecule FRB-CFP / FRB-YFP shows that the CFP and YFP fluorescence mainly aggregates at both ends of Escherichia coli, which is consistent with the theory. The assembly effect of intracellular FRB-CFP / YFP-FRB in the control group without rapamycin was observed by a laser scanning confocal microscope. Figure 5 As shown, the CFP and YFP are evenly dispersed in the Escherichia coli cells without aggregation, which is consistent with the theory. Figure 5 As can be seen, the CFP and YFP are evenly dispersed in the Escherichia coli cells without aggregation, which is consistent with the theory.

[0068] Subsequently, 1.5 μL of the bacterial solution was taken for sample preparation. Under the excitation of a wavelength of 430 nm using a fluorescence lifetime imaging system, the fluorescence lifetime of the CFP protein was detected using a 500 nm ± 7.5 nm filter. The energy transfer phenomenon between the CFP and YFP of the fluorescent proteins was detected by Film Fret. Figure 6 This is for the fluorescence lifetime imaging experiment to detect the fluorescence lifetime of CFP. The results show that the fluorescence lifetime of CFP in the experimental group is significantly reduced, indicating that Fret phenomenon occurs, indicating a relatively high concentration of aggregated proteins. Fret phenomenon occurs only when the distance between the fluorescent protein pairs is less than 10 nm, thus proving that Mi3 chem successfully recruits the cargo protein with an FRB tag, while the FRB-CFP / YFP-FEB in the control group is evenly dispersed throughout the Escherichia coli cells without aggregation. The results further show that the FRB-CFP / YFP-FEB in the experimental group is chem assembled together by the viroid particle Mi3 and aggregates at both ends of Escherichia coli, indicating that the assembly method of the present invention also has a good assembly effect in Escherichia coli cells.

[0069] Finally, it should be noted that although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for constructing a controllable protein recruitment system based on artificial protein cages, characterized in that, It mainly includes the following steps: (1) Connect the gene fragment of the Mi3 subunit of the protein cage with the gene fragment of the FK506-binding protein through a flexible linker sequence to obtain a recombinant gene fragment capable of expressing FKBP-Mi3, insert it into a plasmid vector, and construct a plasmid capable of expressing the protein cage Mi3 in host cells chem , transform it into host cells, culture and induce expression, and then isolate and purify to obtain the protein cage Mi3 chem ; (2) Connect the gene fragment of the FRB domain with the gene fragment of the target protein through a flexible linker sequence to obtain a recombinant gene fragment capable of expressing the target protein molecule with the FRB domain, insert it into a plasmid vector, construct a plasmid capable of expressing the target protein molecule with the FRB domain in a host cell, transform it into the host cell, culture and induce expression, and then isolate and purify to obtain the target protein molecule with the FRB domain; (3) Mix the protein cage Mi3 obtained in step (1) chem with the target protein molecule with an FRB domain obtained in step (2), then add 2 - 20 μM rapamycin for controllable recruitment, and obtain a controllable protein recruitment system by size exclusion chromatography.

2. The construction method according to claim 1, characterized in that The amino acid sequence of the Mi3 subunit of the protein cage in step (1) is positions 133-337 of SEQ ID NO.1, and the amino acid sequence of the FK506 binding protein is positions 14-120 of SEQ ID NO.1; the amino acid sequence encoded by the flexible linker sequence is (GGS) n or (GGGGS) n , where n = 1-10.

3. The construction method according to claim 1, characterized in that The host cells described in step (1) include Escherichia coli, yeast, and animal cells; the plasmid vector is a vector with a T7 promoter, and the gene of the protein cage Mi3 chem is inserted after the T7 promoter of the plasmid vector.

4. The construction method according to claim 1, characterized in that, The target proteins described in step (2) include fluorescent proteins mCherry, CFP, and YFP; the amino acid sequence of mCherry is as shown at positions 14-248 in SEQ ID NO.2; the nucleotide sequence of CFP is as shown at positions 14-252 in SEQ ID NO.3; the nucleotide sequence of YFP is as shown at positions 127-363 in SEQ ID NO.4; the FRB domain can specifically bind to the FK506 binding protein, and the amino acid sequence of FRB is the amino acid sequence encoded by the flexible linker sequence at positions 261-355 in SEQ ID NO.2, and the flexible linker sequence is (GGS) n or (GGGGS) n , where n = 1-10.

5. The construction method according to claim 1, characterized in that, The host cells described in step (2) include Escherichia coli, yeast, and animal cells.

6. The construction method according to claim 1, characterized in that The plasmid described in step (2) is a vector with a T7 promoter, and the gene of the target protein molecule with the FRB domain is inserted after the T7 promoter of the vector.

7. The construction method according to claim 1, wherein The protein cage Mi3 in step (3) chem The molar ratio with the target protein molecule with an FRB domain is 1:1 to 1:3; the concentration of the rapamycin is 2 - 20 μM.

8. A controllable protein recruitment system obtained by the construction method according to any one of claims 1-7.

9. A method for constructing the controllable protein recruitment system according to claim 8 in a host cell, characterized in that, It mainly includes the following steps; (1) Select plasmid vectors that can coexist in host cells, and insert the FKBP-Mi3 recombinant gene fragment in which the gene fragment of the Mi3 subunit of the protein cage and the gene fragment of the FK506-binding protein are connected through a flexible linker sequence, and the recombinant gene fragment of the target protein molecule with the FRB domain in which the gene fragment of the FRB domain and the gene fragment of the target protein are connected through a flexible linker sequence; (2) Transform the recombinant plasmid obtained in step (1) into the same host cell to obtain a host cell capable of simultaneously expressing the protein cage Mi3 chem and the target protein molecule with the FRB domain; (3) Culture the host cells obtained in step (2) and induce the expression of the protein cage Mi3 chem With the target protein molecule carrying the FRB domain, after adding rapamycin, a controllable protein recruitment system is constructed in the host cells.

10. The method according to claim 9, characterized in that, In step (1), the FKBP-Mi3 recombinant gene fragment and the recombinant gene fragment of the target protein molecule with the FRB domain are respectively inserted into coexisting pET series vectors, Duet series vectors, or pBAD series vectors.

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