Plug-and-play Mi3 protein nanocage fusion protein with VNP6 tag and preparation method and application thereof

Abstract

The invention provides a plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag and a preparation method and application of the plug-and-play Mi3 protein nanocage fusion protein, the fusion protein comprises the VNP6 tag, a SpyCatch003 component, a Mi3 protein nanocage and a functional protein sequence, and the VNP6 tag is located at the N end of the fusion protein. The VNP6 peptide sequence or the variant thereof is utilized to induce a vesicle or vesicle-like structure beneficial to protein folding and stabilization in escherichia coli, so that the correct folding rate and activity of recombinant protein are improved while the overall intracellular molecular crowding effect is relieved. The core idea of the invention lies in that the internal structure of cells is regulated at low temperature, and the internal microenvironment of an escherichia coli expression system is optimized, so that protein molecules under high expression load can obtain more reasonable spatial distribution, and the problems of misfolding and aggregation caused by molecular crowding are reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag, and a preparation method and application thereof. Background Art

[0002] In recent years, with the increasing importance of recombinant proteins in biomedicine, industrial production, and basic research, various expression systems, such as mammalian cells, yeast, and Escherichia coli, have been widely developed and utilized. Among them, Escherichia coli has become the preferred platform for industrial-grade recombinant protein production due to its advantages such as short culture cycle, low cost, fast expression speed, and simple operation. However, since Escherichia coli is a prokaryotic organism, its intracellular biosynthetic environment is significantly different from that of eukaryotic cells. Especially when the cell density is high and the expression level is fast, it is easy to cause problems such as abnormal protein folding, nonspecific aggregation, and inclusion body formation. These phenomena are closely related to intracellular molecular crowding. That is, under the competition of high-concentration proteins and other biomacromolecules, the space between molecules is limited, which affects the correct folding and efficient function of proteins, thereby reducing the activity and yield of the final product.

[0003] Traditionally, studies have reported using VNP6 peptide sequences to promote the production of extracellular vesicles to enhance the expression efficiency and secretion levels of specific recombinant proteins. The VNP6 tag can regulate cell signaling and vesicle secretion in some eukaryotic cells, enhancing the extracellular distribution and stability of proteins by inducing the exocytic pathway. However, this strategy primarily focuses on using VNP6 to trigger the activation of cellular protein secretion mechanisms. Its research and application have been concentrated in cell systems capable of complex secretion regulation, while insufficient attention has been paid to a key issue facing prokaryotic microorganisms such as Escherichia coli: molecular crowding caused by the limited internal space within the cell.

[0004] In E. coli, high-density protein expression is often accompanied by the formation of organelle-like or vesicle-like structures, but these structures are not necessarily conducive to the correct folding and function of recombinant proteins. To overcome this bottleneck, it is urgent to develop more innovative strategies to alleviate molecular crowding by regulating the internal microenvironment of the cell. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag, as well as its preparation method and application. This invention improves the "plug-and-play" Mi3 protein nanocage expression system by adding a VNP6 tag to the N-terminus of the "plug-and-play" Mi3 protein nanocage. This peptide sequence promotes intracellular vesicle concentration, effectively alleviating molecular crowding and increasing recombinant protein expression yield.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag, wherein the fusion protein comprises a VNP6 tag, a SpyCatcher003 component, a Mi3 protein nanocage and a functional protein sequence, and the VNP6 tag is located at the N-terminus of the fusion protein.

[0008] The plug-and-play Mi3 protein nanocages described in this paper are a highly thermostable protein nanoparticle platform with excellent structural stability and modifiability. Using the SpyCatcher003 system, Mi3 protein nanocages can be efficiently covalently coupled to various functional proteins to form composite nanoparticles with regular structures. Combining Mi3 protein with a VNP6 tagging strategy not only provides additional support for protein activity regulation during late-stage expression, but also enables the system to achieve higher expression and processing efficiency in the complex biochemical environment within Escherichia coli cells.

[0009] The "plug-and-play" nature of the fusion protein in this invention allows researchers to easily add different functional modules to the Mi3 protein cage, improving experimental flexibility and efficiency, and providing a key tool for protein engineering, drug delivery, and biocatalysis. Through precise molecular design and bioorthogonal chemistry, this invention achieves modular integration and optimization of complex biological functions.

[0010] In the present invention, the amino acids of the VNP6 tag are shown in SEQ ID NO: 3. The VNP6 peptide sequence can be used to enhance the expression efficiency and secretion level of recombinant proteins, mainly by promoting the formation of extracellular vesicles. In eukaryotic cells, the VNP6 tag can regulate cell signal transduction and vesicle secretion mechanisms, and enhance the extracellular distribution and stability of proteins by activating the exocrine pathway. However, existing research strategies mainly focus on how VNP6 activates the protein secretion mechanism of cells, and the research subjects are also limited to cell systems with complex secretion regulation capabilities. The present invention utilizes the VNP6 tag and uses low-temperature induced expression to induce the formation of vesicle structures in cells that are conducive to protein folding and stability, thereby alleviating the molecular crowding effect, in order to increase the expression yield of different mi3 protein nanocages. VNP6 was originally designed to promote extracellular vesicle secretion. By using this method, we increased the protein expression levels of different mi3 protein nanocages by at least 3-5 times.

[0011] SEQ ID NO:3: MDVFKKGFSIADEGVVGAVEKTDQGVTEAAEKTKEGV.

[0012] The plug-and-play Mi3 protein nanocage protein expression system proposed in the present invention, which is based on the VNP6 tag and regulates the formation of intracellular vesicles at low temperature, effectively alleviates the problem of intracellular molecular crowding by inducing internal structural changes, and provides a new technical path for improving the expression level, correct folding and supramolecular structure formation of target proteins.

[0013] Preferably, the functional protein sequence is selected from any one or a combination of at least two of a Sortase A recognition peptide sequence, a Halotag7 protein tag or a snoopcatcher protein tag.

[0014] In the present invention, the functional protein sequence is, for example, selected from the OAAEP1b recognition peptide sequence (NGL) or the connectase recognition peptide sequence (KDPGA).

[0015] In the present invention, the Sortase A recognition peptide sequence (LPXTG) is a specific transpeptidase recognition tag that can be specifically recognized and cleaved by the bacterial Sortase A enzyme, forming an acyl-enzyme intermediate, which then undergoes a transpeptidation reaction with a substrate containing an N-terminal glycine. This recognition sequence is widely used in biotechnology for site-specific protein labeling, protein-protein coupling, antibody modification, and cell surface protein engineering, enabling precise and controllable covalent modification of proteins.

[0016] The Halotag7 protein tag described in this invention is an engineered protein derived from a bacterial dehalogenase that forms highly specific, irreversible covalent bonds with specific halogenated alkane ligands, requiring no additional factors and reacting rapidly under physiological conditions. This versatile tagging system can be applied to protein fluorescence imaging, affinity purification, interaction analysis, and targeted degradation. Because it lacks homologous proteins in mammalian cells, it avoids background interference, making it an important tool in biomedical research.

[0017] In the present invention, the amino acid sequence of the snoopcatcher protein tag is shown in SEQ ID NO: 8:

[0018] GPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDG KYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPATYEFTNGKHY ITNEPIPPK.

[0019] Preferably, the fusion protein comprises a VNP6 tag, a SpyCatcher003 component, a Mi3 protein nanocage and a Sortase A recognition peptide sequence connected in sequence.

[0020] In a specific embodiment of the present invention, the fusion protein includes a VNP6 tag, a SpyCatcher003 component, a Mi3 protein nanocage and a Sortase A recognition peptide sequence connected sequentially from the N-terminus to the C-terminus.

[0021] In the present invention, except for the N-terminal VNP6 tag, all other components and their arrangements can be modified, including the SpyCatcher003 component, Mi3 protein nanocage, and Sortase A recognition peptide sequence. This is because the VNP6 tag acts as a signal peptide-like sequence.

[0022] In the present invention, the SpyCatcher003 component is a component from the SpyTag / SpyCatcher system, which can form a covalent bond with the target protein labeled by SpyTag.

[0023] In the present invention, the Mi3 protein nanocage is a self-assembling protein nanocage derived from a thermostable enzyme and has excellent thermal stability and assembly properties.

[0024] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 1.

[0025] Preferably, the fusion protein comprises a VNP6 tag, a SpyCatcher003 component, a Mi3 protein nanocage, a Halotag7 protein tag and a Sortase A recognition peptide sequence connected in sequence.

[0026] In a specific embodiment of the present invention, the fusion protein includes a VNP6 tag, a SpyCatcher003 component, a Mi3 protein nanocage, a Halotag7 protein tag and a Sortase A recognition peptide sequence connected sequentially from the N-terminus to the C-terminus.

[0027] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 2.

[0028] In a second aspect, the present invention provides a nucleic acid molecule encoding the plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag as described in the first aspect.

[0029] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect.

[0030] In a fourth aspect, the present invention provides a recombinant cell, comprising at least one copy of the expression vector described in the third aspect, or the nucleic acid molecule described in the second aspect is integrated into the genome of the recombinant cell.

[0031] Preferably, the recombinant cell is Escherichia coli.

[0032] In a fifth aspect, the present invention provides a method for preparing the plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag as described in the first aspect, the preparation method comprising: culturing the recombinant cells as described in the fourth aspect, inducing expression with IPTG, and collecting the induced cells; and obtaining the target protein by ammonium sulfate precipitation, anion exchange chromatography, and gel filtration chromatography purification.

[0033] Preferably, the conditions for culturing the recombinant cells are as follows: culturing the recombinant cell monoclonal colonies transferred with the expression vector in a culture medium at a temperature of 35-40°C, and waiting for the bacterial solution OD 600 When the pH value is 0.6-0.8, IPTG is added to induce expression.

[0034] The temperature of 35-40°C can be, for example, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C; OD 600 It is 0.6-0.8, for example, it can be 0.6, 0.7 or 0.8.

[0035] Preferably, the conditions for inducing expression are: a final IPTG concentration of 0.1-0.5 mM, inducing expression for 12-16 h, and the IPTG-induced expression is carried out at a low temperature of 14-18°C.

[0036] The final IPTG concentration is 0.1-0.5 mM, for example, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM or 0.5 mM; 12-16 h can be, for example, 12 h, 13 h, 14 h, 15 h or 16 h; 14-18 ° C can be, for example, 14 ° C, 15 ° C, 16 ° C, 17 ° C or 18 ° C.

[0037] In the present invention, expression is performed in recombinant cells (Escherichia coli) using low-temperature culture conditions (e.g., 16° C.). Under these conditions, the secretion of extracellular vesicles is not promoted, but the concentration of intracellular vesicles is beneficial, thereby preventing intracellular molecular crowding.

[0038] In the present invention, the steps of ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography purification include:

[0039] (1) Salting-out method: Cell lysate containing recombinant VNP6-SpyCatcher003-Mi3 nanocages was incubated with 10-50% (mass-volume ratio) ammonium sulfate for 1-24 hours to promote protein precipitation.

[0040] (2) Centrifugal collection: The precipitated protein was collected by centrifugation or filtration, and the precipitate was then resuspended in phosphate-buffered saline (PBS) buffer.

[0041] (3) Anion exchange chromatography: The filtered protein solution was loaded onto an anion exchange chromatography column connected to a fast protein liquid chromatography system for further purification, and the target protein was eluted using a linear gradient from PBS to 1-3 M sodium chloride.

[0042] (4) Desalting: Desalting is performed using a desalting column, or gel filtration chromatography: size exclusion chromatography (SEC) purification is performed on a fast protein liquid chromatography system to obtain a high-purity target protein.

[0043] This invention has devised a promising new protein expression strategy, utilizing VNP6 peptide sequences or variants thereof to induce the formation of vesicles or vesicle-like structures within Escherichia coli that are conducive to protein folding and stabilization. This, in turn, reduces the overall intracellular molecular crowding while improving the correct folding rate and activity of recombinant proteins. Unlike the traditional use of VNP6 to promote extracellular vesicle secretion, the core concept of this invention is to regulate the internal cell structure through low temperatures and optimize the microenvironment within the E. coli expression system, thereby achieving a more rational spatial distribution of protein molecules under high expression loads and reducing the problems of misfolding and aggregation caused by molecular crowding.

[0044] In the present invention, comparative experiments showed that the expression yield of Escherichia coli expressing VNP6-SpyCatcher003-Mi3-LPETGGH and VNP6-SpyCatcher003-Mi3-Halotag7-LPETGGH constructs was significantly higher than that of cells expressing only SpyCatcher003-Mi3-LPETGGH and SpyCatcher003-Mi3-Halotag7-LPETGGH constructs. At the same time, the in vivo supramolecular assembly to form nanocages, gel filtration chromatography, dynamic light scattering and transmission electron microscopy detection proved that the plug-and-play Mi3 protein nanocage containing the VNP6 tag maintained the supramolecular structure of the Mi3 protein nanocage.

[0045] In the present invention, the plug-and-play Mi3 protein nanocage fusion protein containing a VNP6 tag can be modified through a one-pot dual-functionalization strategy, that is, using an orthogonal ligation reaction mediated by SpyTag003 / SpyCatcher003 and Sortase A, a fluorescent peptide (such as GGRSYK(FAM)-NH2) or a fluorescent protein (such as GGG-SpyCatcher003-mNG3A) and a targeting molecule (such as ZHER2:342-SpyTag003) are simultaneously connected to the same nanocage, thereby realizing the efficient preparation of multifunctional nanomaterials.

[0046] In the sixth aspect, the present invention provides the application of the preparation method of the plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag as described in the first aspect, the nucleic acid molecule as described in the second aspect, the expression vector as described in the third aspect, the recombinant cell as described in the fourth aspect, or the plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag as described in the fifth aspect, and the application includes: assembling vaccine antigen nanoparticles, constructing a multi-enzyme cascade reaction system, developing a biosensor platform based on protein nanocages, or preparing a drug delivery system.

[0047] In the present invention, the application in assembling vaccine antigen nanoparticles: VNP6-tagged Mi3 protein nanocages can serve as an ideal vaccine carrier platform. Through plug-and-play technology, multiple antigen molecules can be displayed in a high-density and orderly manner on the surface of the nanocage to form a multivalent antigen display system. This design can significantly enhance the cross-linking effect of B cell receptors and increase the intensity of humoral immune response; at the same time, different types of antigens or different epitopes of the same antigen can be displayed to induce a wider range of immune protection. The nanoparticle size (about 20-25nm) is suitable for efficient uptake by antigen-presenting cells, enhancing T cell responses, and its structure can protect antigens from degradation in the body and prolong antigen exposure time.

[0048] In the present invention, the application in constructing a multi-enzyme cascade reaction system: VNP6-tagged Mi3 protein nanocages can provide an ideal scaffold platform for multi-enzyme cascade reactions, realizing the directional and controllable assembly of different enzyme molecules. Immobilizing multiple enzymes of the cascade reaction on the surface or inner cavity of the nanocage can significantly shorten the diffusion distance of the reaction intermediates and improve the reaction efficiency by 5-20 times; the unique microenvironment formed in the inner cavity of the nanocage can provide optimal conditions for enzyme catalysis, realize the efficient transfer of intermediates, and reduce side reactions. This system protects the immobilized enzymes from environmental factors and prolongs the catalytic life; at the same time, by adjusting the loading ratio of different enzymes, the efficiency of complex biochemical transformation pathways is optimized. This nanoenzyme system can be widely used in the green synthesis of high-value products such as fine chemicals, drug precursors, and biofuels, providing an innovative solution for industrial biocatalysis.

[0049] In the present invention, in the development of a biosensor platform based on protein nanocages, the VNP6-tagged Mi3 protein nanocage can be used as an excellent platform for constructing a highly sensitive biosensor. A single nanocage can integrate multiple signal molecules (such as fluorophores, electrochemically active molecules) to achieve signal amplification and significantly improve detection sensitivity; at the same time, it can be loaded with different types of detection elements to achieve multimodal signal output such as fluorescence, electrochemistry, and colorimetry. By integrating high-affinity recognition elements (such as antibody fragments and aptamers), the detection specificity is improved, and the integration of recognition, transduction, and signal amplification functions is achieved on a single nanoparticle. This system can construct a sensor network with signal processing capabilities by designing the interactions between nanocages. It is suitable for highly sensitive real-time monitoring of biomarkers, environmental pollutants, or food safety risk factors in the body, providing a new solution for biodetection technology.

[0050] In the present invention, the VNP6-tagged Mi3 protein nanocage is used in the preparation of a drug delivery system, which has significant advantages as a drug delivery carrier. Its inner cavity can efficiently load small molecule drugs, nucleic acid drugs or protein drugs, and its outer surface can be connected to a targeting ligand to achieve precise delivery to specific tissues or cell types. The system can integrate linkers that are sensitive to pH, temperature, and redox environment to achieve selective release of drugs at specific sites; a single nanocage can be loaded with multiple drugs at the same time to achieve a synergistic therapeutic effect. Through targeted delivery, the systemic exposure of drugs is reduced, toxic side effects are significantly reduced, and at the same time, the drugs are protected from enzymatic degradation in the body, thereby improving bioavailability and therapeutic effects, providing key technical support for precision medicine.

[0051] The numerical range described in the present invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The present invention not only significantly improves the protein expression yield through the VNP6 tag-mediated intracellular vesicle concentration process, but also completely preserves the supramolecular assembly characteristics of the Mi3 protein nanocage and the biological activity of each functional module, laying the foundation for the construction of a highly efficient multifunctional protein nanoplatform.

[0054] (2) The VNP6 tag specifically promotes intracellular vesicle concentration under low temperature conditions, significantly alleviating molecular crowding; compared with the control without the VNP6 tag, the expression yield is increased by 300-500%.

[0055] (3) The purification process used in the present invention is simplified, does not require affinity chromatography, and is more suitable for large-scale production.

[0056] (4) The fusion protein of the present invention maintains the supramolecular assembly function and structural stability of the Mi3 nanocage. At the same time, it can be diversified and modified and its application expanded by replacing the C-terminal functional elements.

[0057] (5) The present invention innovatively achieves one-pot bifunctionalization modification. By using the orthogonal ligation reaction mediated by SpyTag003 / SpyCatcher003 and Sortase A, peptides / proteins (such as GGRSYK(FAM)-NH2 or GGG-SpyCatcher003-mNG3A) and targeting molecules (such as ZHER2:342-SpyTag003) can be simultaneously connected to the same nanocage, simplifying the production process and expanding the application functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a comparative analysis of the SDS-PAGE expression levels of the recombinant protein SpyCatcher003-Mi3-LPETGGH with and without the VNP6 tag.

[0059] Figure 2 This is a comparative analysis of the SDS-PAGE expression levels of the recombinant proteins SpyCatcher003-Mi3-Halotag7-LPETGGH with and without the VNP6 tag.

[0060] Figure 3 This is the purification result of VNP6-SpyCatcher003-Mi3-LPETGGH protein.

[0061] Figure 4 This is the purification result of VNP6-SpyCatcher003-Mi3-Halotag7-LPETGGH protein.

[0062] Figure 5 Dynamic light scattering (DLS) analysis and electron microscopy images of VNP6-SpyCatcher003-Mi3-LPETGGH.

[0063] Figure 6 This is the dynamic light scattering (DLS) analysis result of VNP6-SpyCatcher003-Mi3-Halotag7-LPETGGH.

[0064] Figure 7 This is the result of functional modification of VNP6-SpyCatcher003-Mi3-LPETGGH with ZHER2:342-SpyTag003 or Zwt:342-SpyTag003 using SpyTag003 / SpyCatcher003 connection technology.

[0065] Figure 8 The results are the functionalization modification of VNP6-SpyCatcher003-Mi3-LPETGGH with fluorescent peptide (GGYSK(Biotin)K(FAM)-NH2) using Sortase A-mediated ligation reaction.

[0066] Figure 9 The results are the result of one-pot bifunctionalization modification of VNP6-SpyCatcher003-Mi3-LPETGGH using SpyTag003 / SpyCatcher003 and Sortase A-mediated ligation reaction, and simultaneous ligation of fluorescent peptide (GGRSYK(FAM)-NH2) and ZHER2:342-SpyTag003.

[0067] Figure 10 The results are from a one-pot dual-functionalization modification of VNP6-SpyCatcher003-Mi3-LPETGGH using SpyTag003 / SpyCatcher003 and Sortase A-mediated ligation reaction, which simultaneously linked fluorescent protein (GGG-SpyCatcher003-mNG3A) and ZHER2:342-SpyTag003. DETAILED DESCRIPTION

[0068] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0069] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0070] Example 1

[0071] The constructs VNP6-SpyCatcher003-Mi3-LPETGGH and VNP6-SpyCatcher003-Mi3-Halotag7-LPETGGH were prepared.

[0072] The construct consists of the following functional elements:

[0073] (1) VNP6 tag: located at the N-terminus, used to induce the formation and concentration of intracellular vesicles.

[0074] (2) SpyCatcher003: A component from the SpyTag / SpyCatcher system that can form a covalent bond with the SpyTag-tagged target protein.

[0075] (3) Mi3 protein nanocages: self-assembling protein nanocages derived from thermostable enzymes with excellent thermal stability and assembly properties.

[0076] (4) LPETGGH: Sortase A recognition sequence, used for enzymatic labeling reaction.

[0077] (5) Halotag7 (optional): a protein tag used to covalently link small molecule ligands.

[0078] VNP6-SpyCatcher003-Mi3-LPETGGH protein coding sequence SEQ ID NO: 1.

[0079] MDVFKKGFSIADEGVVGAVEKTDQGVTEAAEKTKEGVMSGGGSGDYDIPTTENLYFQSIGGGSSVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGGSGGSGGSGGSMKMEELFKKHKIVAVLRAN SVEEAKKKALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMK LGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTEGSGEPEAGSGSGSLPETGGH.

[0080] VNP6-SpyCatcher003-Mi3-Halotag7-LPETGGH protein coding sequence SEQ ID NO: 2.

[0081] MDVFKKGFSIADEGVVGAVEKTDQGVTEAAEKTKEGVMSGGGSGDYDIPTTENLYFQSIGGGSVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGGSGGSGGSGGSMKMEELFKKHKIVAVLRANSVEEAKKKALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTEGSGEPEAGSGSGSGGSGGSGGSGGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGSGSGSLPETGGH。

[0082] The amino acid sequence of the VNP6 tag, SEQ ID NO:3.

[0083] MDVFKKGFSIADEGVVGAVEKTDQGVTEAAEKTKEGV.

[0084] The amino acid sequence of SpyCatcher003, SEQ ID NO:4.

[0085] SIGGGSVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATM ELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNED GQVTVDGEATEGDAHT.

[0086] The amino acid sequence of Mi3 protein nanocage is SEQ ID NO: 5.

[0087] GGSGGSGGSGGSMKMEELFKKHKIVAVLRANSVEEAKKKALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEK GVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTEGSGEPEAGSGSGS.

[0088] The amino acid sequence of the Sortase A recognition sequence is SEQ ID NO: 6. LPETGGH.

[0089] The amino acid sequence of Halotag7 is SEQ ID NO: 7.

[0090] EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETF QAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEIS.

[0091] Example 2

[0092] Construct expression

[0093] The coding sequence of the above construct was cloned into a vector suitable for expression in E. coli and transformed into BL21 (DE3) or other suitable expression strains. The expression steps are as follows:

[0094] 1. Inoculate the transformed strain into LB medium containing appropriate antibiotics and culture at 37°C with shaking until the OD 600 Reach 0.6-0.8.

[0095] 2. Cool down to 16°C and add IPTG to a final concentration of 0.1-0.5 mM to induce expression.

[0096] 3. Continue culturing at 16°C for 12-16 hours.

[0097] 4. Collect the cells by centrifugation and crush them by ultrasound or pressure to obtain cell lysate.

[0098] 5. Purify the target protein by ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography without affinity chromatography.

[0099] SDS-PAGE electrophoresis analysis results ( Figure 1 and Figure 2 ) clearly showed that the expression level of the recombinant proteins with VNP6 tags (VNP6-SpyCatcher003-Mi3-LPETGGH and VNP6-SpyCatcher003-Mi3-Halotag7-LPETGGH) in Escherichia coli cells was significantly increased. Under exactly the same culture conditions and induction parameters, compared with the control group without VNP6 tags (SpyCatcher003-Mi3-LPETGGH and SpyCatcher003-Mi3-Halotag7-LPETGGH), the expression level of the experimental group proteins with VNP6 tags was significantly increased. This result fully confirms the core mechanism of the VNP6 tag in the present invention that it effectively alleviates molecular crowding by promoting the concentration of intracellular vesicles, thereby improving the expression level of the target protein.

[0100] Example 3

[0101] Protein purification process

[0102] This example develops an efficient protein purification process suitable for industrial scale-up, which includes the following three main steps:

[0103] (1) Ammonium sulfate fractional precipitation removes most of the impurity proteins;

[0104] The cell lysate containing the recombinant VNP6-SpyCatcher003-Mi3 nanocages was incubated with 15% (mass-volume ratio) ammonium sulfate at 4°C for 1 hour, during which the mixture was rotated at 100 rpm to promote protein precipitation.

[0105] The precipitated protein was collected by centrifugation (10000 g, 20 min, 4°C), and the precipitate was then resuspended in PBS buffer and filtered using a 0.22 μm syringe filter to remove insoluble impurities.

[0106] (2) Anion exchange chromatography further removes DNA and negatively charged impurities;

[0107] The filtered protein solution was loaded onto an anion exchange chromatography column connected to a fast protein liquid chromatography system for further purification, and the target protein was eluted using a linear gradient from PBS to 1 M sodium chloride.

[0108] (3) Final purification was achieved by gel filtration chromatography.

[0109] Desalt the protein using a desalting column or perform gel filtration chromatography: Purify the protein by size exclusion chromatography (SEC) at a flow rate of 0.3 mL / min on a fast protein liquid chromatography system to obtain a highly pure target protein.

[0110] This purification process does not require the use of expensive affinity chromatography materials, and each step has high yield and good reproducibility ( Figure 3 and Figure 4 )

[0111] Example 4

[0112] Characterization and validation

[0113] To verify whether the in vivo vesicle concentration process preserves the assembly state and coupling reaction efficiency of the protein nanocages composed of VNP6-SpyCatcher003-Mi3-LPETGGH and VNP6-SpyCatcher003-Mi3-Halotag7-LPETGGH proteins, we first characterized the assembly state of the protein nanocages using purified VNP6-SpyCatcher003-Mi3-LPETGGH and VNP6-SpyCatcher003-Mi3-Halotag7-LPETGGH proteins.

[0114] Dynamic light scattering (DLS) analysis results showed that ( Figure 5), VNP6-SpyCatcher003-Mi3-LPETGGH formed a monodisperse particle size distribution with an average hydrodynamic radius of 37.94 nm, indicating that the sample has excellent uniformity. The average hydrodynamic radius of VNP6-SpyCatcher003-Mi3-Halotag7-LPETGGH was slightly larger, at 40.21±0.82 nm, which is consistent with the increase in molecular size caused by the introduction of Halotag7, while still maintaining good supramolecular assembly properties ( Figure 6 ).

[0115] Transmission electron microscopy (TEM) negative staining observation further confirmed the above results ( Figure 5 ), clearly demonstrating the uniform, regular cage-like structure formed by VNP6-SpyCatcher003-Mi3-LPETGGH, with an average diameter of 22.6±2.2 nm. Electron microscopy images revealed a typical spherical structure of the nanocages, with a regular polyhedral surface morphology, uniform interparticle spacing, and no apparent aggregation, demonstrating that the VNP6 tag does not affect the inherent self-assembly properties of the Mi3 protein.

[0116] Example 5

[0117] Functional feature verification

[0118] To verify that the VNP6-tagged fusion protein maintained its original functional properties, the following reaction tests were performed:

[0119] 1. Coupling reaction with SpyTag003-labeled target protein: Purified VNP6-SpyCatcher003-Mi3-LPETGGH was mixed with ZHER2:342-SpyTag003. SDS-PAGE confirmed that the function of SpyCatcher003 was fully retained. Figure 7 )

[0120] 2. Site-specific labeling reaction mediated by Sortase A: The C-terminal LPETGGH sequence of VNP6-SpyCatcher003-Mi3-LPETGGH can be effectively recognized by Sortase A. After reacting with a fluorescent peptide containing a GG-sequence (such as GGYSK(Biotin)K(FAM)-NH2) substrate, it was confirmed that the C-terminal functional element has good activity. Figure 8 )

[0121] 3. One-pot bifunctionalization of VNP6-SpyCatcher003-Mi3-LPETGGH: Using SpyTag003 / SpyCatcher003 and Sortase A-mediated ligation reaction, VNP6-SpyCatcher003-Mi3-LPETGGH was reacted with a fluorescent peptide (e.g., GGRSYK(FAM)-NH2) or protein (e.g., GGG-SpyCatcher003-mNG3A) substrate containing a GG-sequence and ZHER2:342-SpyTag003. Figure 9 and Figure 10 )

[0122] The above verification results fully demonstrate that the intracellular vesicle concentration process mediated by the VNP6 tag not only significantly improves the protein expression yield, but also completely retains the supramolecular assembly characteristics of the Mi3 protein nanocage and the biological activity of each functional module, laying the foundation for the construction of a highly efficient multifunctional protein nanoplatform.

[0123] In summary, the present invention successfully solves the molecular crowding problem under high-density culture of E. coli through VNP6 tagging and low-temperature regulation, providing a new efficient and economical strategy for the production of industrial-grade recombinant protein nanocages.

[0124] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag, characterized in that: The fusion protein includes a VNP6 tag, a SpyCatcher003 component, a Mi3 protein nanocage and a functional protein sequence, and the VNP6 tag is located at the N-terminus of the fusion protein.

2. The plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag according to claim 1, characterized in that: The functional protein sequence is selected from any one or a combination of at least two of a Sortase A recognition peptide sequence, a Halotag7 protein tag or a snoopcatcher protein tag.

3. The plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag according to claim 1 or 2, characterized in that: The fusion protein includes a VNP6 tag, a SpyCatcher003 component, a Mi3 protein nanocage, and a Sortase A recognition peptide sequence connected in sequence; Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO:

1.

4. The plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag according to any one of claims 1-3, characterized in that: The fusion protein includes a VNP6 tag, a SpyCatcher003 component, a Mi3 protein nanocage, a Halotag7 protein tag and a Sortase A recognition peptide sequence connected in sequence; Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO:

2.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag according to any one of claims 1 to 4.

6. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to claim 5.

7. A recombinant cell, characterized in that The recombinant cell contains at least one copy of the expression vector according to claim 6, or the nucleic acid molecule according to claim 5 is integrated into the genome of the recombinant cell.

8. A method for preparing a plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag according to any one of claims 1 to 4, characterized in that: The preparation method comprises: culturing the recombinant cell according to claim 7, inducing expression with IPTG, collecting the induced cells; and purifying by ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography to obtain the target protein.

9. The method for preparing a plug-and-play Mi3 protein nanocage fusion protein with a VNP6 tag according to claim 8, characterized in that: The conditions for culturing the recombinant cells are as follows: culturing the recombinant cell monoclonal colonies transferred with the expression vector in a culture medium at a temperature of 35-40° C. and waiting for the bacterial solution OD 600 When the pH value is 0.6-0.8, IPTG is added to induce expression; Preferably, the conditions for inducing expression are: a final IPTG concentration of 0.1-0.5 mM, inducing expression for 12-16 h, and the IPTG-induced expression is carried out at a low temperature of 14-18°C.

10. Use of the method for preparing the VNP6-tagged plug-and-play Mi3 protein nanocage fusion protein according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the recombinant cell according to claim 7, or the VNP6-tagged plug-and-play Mi3 protein nanocage fusion protein according to claim 8, characterized in that: The applications include: assembling vaccine antigen nanoparticles, constructing multi-enzyme cascade reaction systems, developing biosensing platforms based on protein nanocages, or preparing drug delivery systems.

Images