Protein nano double-shell structure commonly used for electron microscope and crystal research and application thereof

Abstract

The invention discloses a protein nanometer double-shell structure capable of being used for electron microscopy and crystal research and a construction method and application thereof, the protein nanometer double-shell structure comprises an inner shell, an outer shell and target protein clamped between the inner shell and the outer shell, the inner shell is ferritin, and the ferritin comprises ferritin from escherichia coli. The constructed protein nano double-shell structure can meet the requirement of an electron microscope technology for protein molecular weight, LRPPRC protein functional domain crystals can be screened out, the reconstruction method based on conformation does not need mutation, the efficiency of obtaining recombinant protein is greatly improved, and meanwhile, the nano structure can maintain the affinity of target protein and nucleic acid aptamers.

Description

Technical Field

[0001] The invention relates to the technical field of structural biology, and in particular to the construction and application of a protein nano double shell structure for electron microscopy and crystal research. Background Art

[0002] The structure of a protein determines its function. The analysis of protein structure not only helps to reveal the complex mechanisms in life processes, but also helps to design and transform proteins with specific functions and characteristics to better meet specific needs. By analyzing the interaction pattern between drugs and target proteins, it can also guide the design and development of drugs. Nucleic acid aptamers are single-stranded oligonucleotides separated from random DNA or RNA libraries through exponential enrichment of ligand system evolution technology. They can be folded into unique spatial structures and bind to corresponding targets including proteins, ions, and even entire cells with high affinity and high specificity. Nucleic acid aptamers are widely used in drug design and development, and delivery coupling. It is of great significance to study the binding of nucleic acid aptamers to protein targets.

[0003] At present, most of the targets of nucleic acid aptamers are proteins of medium molecular weight, and the research methods for nucleic acid aptamer protein targets and interaction patterns are mostly limited to nuclear magnetic resonance spectroscopy. Since magnetic resonance technology is limited to small proteins below 35kD, and has high requirements for the stability of the studied proteins in solution, high purity and concentration, and isotope labeling, many proteins are difficult to resolve. According to literature reports, compared with nuclear magnetic resonance spectroscopy, the other two mainstream structural biology technologies - very powerful electron microscopy reconstruction technology and the most classic X-ray crystallography technology - are rarely used. The important reason is that electron microscopy technology requires large proteins above 300kDa to be easily seen, and X-ray single crystal diffraction technology has the clearest structure, but the ability to grow protein crystals is the first necessary step to restrict this technology, and it is also one of the main bottlenecks.

[0004] LRPPRC protein (leucine-rich pentatricopeptide repeat-containing protein) is a non-small cell lung cancer marker and therapeutic target discovered by nucleic acid aptamer screening. Studies have shown that nucleic acid aptamers bind to the carboxyl terminus of LRPPRC protein, but the structure of the complex interacting with LRPPRC and nucleic acid aptamers has not been studied. Due to the high flexibility of nucleic acids, direct analysis is very difficult, so the starting point is to first analyze the LRPPRC protein structure.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a protein nano double-shell structure method that can be used for electron microscopy and crystal research. The constructed protein nano double-shell structure can meet the requirements of electron microscopy technology for protein molecular weight, and can also screen out LRPPRC protein functional domain crystals. At the same time, the protein nano double-shell structure target protein can still interact with the nucleic acid aptamer.

[0007] In a first aspect, the present invention provides a protein nano double-shell structure for electron microscopy and crystal research, which includes an inner shell, an outer shell, and a target protein sandwiched between the inner and outer shells, wherein the inner shell is ferritin, and the ferritin includes ferritin derived from Escherichia coli.

[0008] In a second aspect, the present invention provides a method for constructing the above-mentioned protein nano double shell, which comprises: introducing an expression vector in which the genes of the tag protein, the target protein and the ferritin are sequentially connected into a host bacterium, culturing and inducing protein expression, and obtaining the protein nano double shell structure after separation and purification.

[0009] In a third aspect, the present invention provides the application of the above-mentioned protein nano double shell structure in electron microscopy and crystal structure research, including:

[0010] The purified protein nano double-shell structure is reconstructed in two dimensions by transmission electron microscopy imaging to confirm that the morphology of the protein nano double-shell structure containing the target protein is successfully constructed; and / or

[0011] The purified protein nano double shell structure is filtered, concentrated and screened to obtain protein crystals of the target protein.

[0012] In a fourth aspect, the present invention also provides a fusion protein comprising: ferritin derived from Escherichia coli, a target protein connected to the N-terminus of the ferritin derived from Escherichia coli, and a tag protein expressed by fusion at the N-terminus of the target protein; the target protein is LRPPRC protein; and the tag protein is maltose binding protein.

[0013] In a fifth aspect, the present invention also provides a vector comprising an expression cassette for expressing the above-mentioned fusion protein.

[0014] In a sixth aspect, the present invention further provides a kit comprising the above-mentioned protein nano double-shell structure, fusion protein or vector.

[0015] The present invention has the following beneficial effects:

[0016] The present invention constructs a protein nano double shell structure for electron microscopy and crystal research by using ferritin from Escherichia coli as the inner shell. The protein nano double shell structure can be commonly used in electron microscopy and crystal structure research, has strong practicality, and the generated nanostructure can maintain the interaction between the target protein and the nucleic acid aptamer. It has a wide range of applications and is not only commonly used in electron microscopy and crystal research, but also has universality, and is suitable for protein segments with similar structural features to the LRPPRC protein (α-helical ending and capable of interacting with nucleic acid aptamers). This idea of ​​fusion expression of target protein and natural multimeric protein based on conformation is extensible, and can make full use of self-assembled multimeric proteins similar to ferritin, so that more target proteins can be seen by electron microscopy or crystallized with the help of the easy crystallization characteristics of natural multimeric proteins, so that the structure can be studied, and this method of protein reconstruction based on a single conformation is more efficient without mutation. The target protein of the present invention is a nucleic acid aptamer target protein, and the segment can still interact with the nucleic acid aptamer after reconstruction. Therefore, the method provided by the present invention can simply and efficiently produce a universal fusion protein for electron microscopy and crystal research and maintain interaction with the original nucleic acid aptamer. This method is of great significance for protein structure research, research on the interaction between nucleic acid aptamers and proteins, and even nucleic acid drug development, and is also popularizable and inspiring for protein structure research. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 This is a structural comparison diagram of mouse ferritin and E. coli ferritin;

[0019] Figure 2 Schematic diagram of the design of LRPPRC fusion protein in the embodiment;

[0020] Figure 3 The results of affinity purification of MBP-tagged proteins and preparation of electron microscopy samples in the examples are shown;

[0021] Figure 4 The results of protein reverse hanging purification and crystal sample preparation screening after cutting off the MBP tag in the examples;

[0022] Figure 5 The results of the verification of the interaction between the fusion protein containing the target protein constructed in the example and the nucleic acid aptamer;

[0023] Figure 6These are the results of affinity purification characterization by other methods in the experimental examples. DETAILED DESCRIPTION

[0024] Ferritin is usually a multimeric protein composed of 24 independent subunits. Its unique protein cage structure and self-assembly properties can effectively load a variety of imaging agents and drug molecules, and therefore it is widely used in in vivo delivery and disease diagnosis and treatment. Ferritin is currently widely studied and used mainly as a drug carrier. A study published in the journal ACS CentralScience in 2022 disclosed the use of ferritin's ability to self-assemble into 24 tetrahedral nanocages. By designing a protein double shell, the target protein KIK domain is sandwiched between ferritin as the inner shell and maltose binding protein as the outer shell, which can help small proteins resolve cryo-electron microscopy structures. However, the shortcoming of this study is that it is necessary to introduce disulfide bond mutations at the interface between the target protein and ferritin to produce a conformationally stable anchor point to form a recombinant protein.

[0025] Based on this study, the inventors found that α-helix is ​​the most stable secondary structure of amino acids. Figure 1 , using Escherichia coli ferritin (PDB: 1BFR) with an alpha helix at the amino end to replace the mouse ferritin (PDB: 3WNW) with a loop irregular coil at the amino end reported in 2022, the carboxyl end of the target protein ending with an alpha helix (such as the LRPPRC protein) can be directly fused and expressed with the amino end of the ferritin without mutation, and the target protein can still have affinity with the aptamer in this double-shell design. Further electron microscopy imaging and two-dimensional reconstruction analysis revealed the spike morphology of the fusion protein, indicating that the recombinant protein construction can be successful and electron microscopy-based imaging can be achieved. In addition, the fusion protein constructed based on ferritin also helps the target protein, which was originally not easy to crystallize, to grow protein crystals, which not only provides the possibility for the analysis of the target protein structure, but also provides a possible strategy for studying the interaction between the target protein and the aptamer based on the structure and developing related aptamer inhibitors based on the interaction.

[0026] Based on the difficulties and shortcomings of the research methods of LRPPRC protein structure and its interaction structure with nucleic acid aptamers, the present invention studies a LRPPRC protein assembly construction strategy that can be universally used for electron microscopy and crystal research, that is, to construct a protein nano double shell structure containing LRPPRC protein. The protein nano double shell structure can be universally used for electron microscopy and crystal research of LRPPRC protein, which can not only meet the requirements of electron microscopy technology for protein molecular weight, but also screen out LRPPRC protein functional domain crystals without mutation, and also meet the requirements of nucleic acid aptamer and target affinity. This method is of great significance for protein structure research, nucleic acid aptamer and protein interaction research, and even nucleic acid drug inhibitor development, and is also popular and inspiring for protein structure research.

[0027] Specifically, a protein nano double shell structure for electron microscopy and crystal research constructed in the present invention is as follows: Figure 2 As shown, it comprises an inner shell, an outer shell and a target protein sandwiched between the inner and outer shells. The inner shell is ferritin, and the ferritin comprises ferritin derived from Escherichia coli (E. coli ferritin).

[0028] In the present invention, the target protein studied is LRPPRC protein, but the construction method of the present invention for electron microscopy and crystal research is not limited to LRPPRC protein, and the object of study can also be other protein segments ending with α helices. If the protein segment is a nucleic acid aptamer target protein, then the target protein can maintain affinity with the nucleic acid aptamer after assembling the nanostructure. Therefore, the protein nano double shell structure of the present invention provides the possibility of studying the structure of the complex of the interaction between the nucleic acid aptamer and the target protein.

[0029] In the present invention, the shell is a label protein. For the selection of label protein, any label protein conventionally used in the art can be used, including maltose binding protein label, His label, Strep label, TAP label, S label, SBP label, Arg label, calmodulin binding peptide label, cellulose binding domain label, DsbA label, c-myc label, glutathione S-transferase label, FLAG label, HAT label, NusA label and thioredoxin label. Experimental results show that when the shell is maltose binding protein (Maltose binding protein, MBP), the effect of the protein nano double shell structure constructed is better. The maltose binding protein here can be maltose binding protein of any source, such as artificial synthesis or recombinant generation, and the present invention is not limited to this.

[0030] The above protein nano double shell structure can be expressed by a fusion protein comprising ferritin from E. coli, a target protein and a tag protein. In the fusion protein, the target protein is connected to the N-terminus of the ferritin, and the tag protein is connected to the N-terminus of the target protein. In some embodiments, the above fusion protein can be expressed as: NH2-tag protein-target protein-ferritin-COOH. In order to successfully construct a fusion protein and study the interaction between the target protein and the nucleic acid aptamer, the present invention selected the 1025-1391 amino acid segment at the end of the α-helix at the carboxyl terminal of the LRPPRC protein and the E. coli ferritin starting from the α-helix at the amino terminal to recombinant and maintain the interaction between the recombinant protein and the nucleic acid aptamer.

[0031] Optionally, the tag protein and the target protein can be connected via a linker. The linker can be a short peptide sequence conventionally used in the art, such as a histidine tag, etc., which will not be described in detail here.

[0032] In the present invention, the method for constructing the above-mentioned protein nano double-shell structure includes: introducing an expression vector in which the genes of the tag protein, the target protein and the ferritin are sequentially connected into a host bacterium, culturing and inducing protein expression, and obtaining the protein nano double-shell structure after separation and purification.

[0033] In some embodiments, the target protein is the 1025-1391 amino acid fragment of the carboxyl terminus of LRPPRC, whose amino acid sequence is shown in SEQ ID NO:1, and the amino acid sequence of ferritin derived from Escherichia coli is shown in SEQ ID NO:2.

[0034] The expression vector and host bacteria used in the present invention can be vectors and strains conventionally used in the art, as long as the vector can express the above fusion protein. In some embodiments of the present invention, the expression vector is pMAL-c2X, which contains the sequence of maltose binding protein. When constructing the vector, the expression cassette containing the fusion of target protein and ferritin is directly constructed into pMAL-c2X, and then transferred into the host bacteria to induce expression to purify the above fusion protein.

[0035] In some embodiments, purification includes: protein tag purification and molecular sieve treatment.

[0036] The tags used in the purification process include: MBP tag and / or His tag; preferably, the MBP-LRPPRC-E.coli ferritin protein obtained by purification with the MBP tag; or, the LRPPRC-E.coli ferritin protein obtained by purification with the MBP tag and purification with the His tag, the effect of purifying the target protein with the MBP tag is better than that with the His tag alone.

[0037] After protein tag purification, in order to obtain a better purification effect, the present invention also performs molecular sieve treatment on the sample. The present invention does not limit the optional purification column in the molecular sieve treatment, and the optional purification column includes HiPrep 16 / 60Sephacryl S-300High Resolution purification column. After comparative analysis, the purification effect of molecular sieve is better than that of ion exchange chromatography, wherein the second peak of the molecular sieve is a correct single state compared to the standard sample marker, and the collected nanostructure has a good effect for electron microscopy analysis and can also be used for growing crystals.

[0038] The above protein nano double shell structure can be used to conduct electron microscopy and crystal research on the LRPPRC protein structure.

[0039] Among them, the electron microscopy structure analysis is: the purified protein nano double shell structure is two-dimensionally reconstructed through transmission electron microscopy imaging to determine the successful assembly of the protein nano double shell and the target protein sandwich structure.

[0040] Crystal research is to obtain the protein crystals of the target protein by filtering, concentrating and screening the purified protein nano double shell structure, thereby screening out the LRPPRC protein functional domain crystals. The optional operation methods are as follows:

[0041] The purified protein nano double shell structure is concentrated with an ultrafiltration tube and replaced with 100mM NaCl, 20mMTris-HCl (pH8.0) buffer, and then the crystals are screened by the sitting drop method. The operation steps are: add 40μL of each screening buffer to the large wells of the 96-well protein crystallization plate in turn, add 1μL of the purified protein nano double shell structure to the small wells next to it, and then transfer 1μL of the corresponding screening buffer from the large wells to the small wells next to it, gently mix the protein nano double shell structure and the screening buffer, and then plastic seal it, place it in a protein crystal incubator at room temperature for one week, and then observe the protein crystals through a microscope. The solvent conditions for the crystallization of the fusion protein of the present invention are Crystal Screen 2 TM Reagent screening solution No. 47: 0.1M bicine (pH 9.0), 2.0MMgCl2.

[0042] The above method can be used to enlarge the functional domain of LRPPRC protein so that it can be characterized by electron microscopy. It can also use the easy crystallization property of ferritin to construct a fusion protein to help the LRPPRC protein that is not easy to crystallize to grow protein crystals for subsequent crystallographic structural research. At the same time, the protein produced by the above method can maintain interaction with nucleic acid aptamers, providing a platform for studying the structure of LRPPRC protein and the interaction structure between protein and nucleic acid aptamers and developing related nucleic acid aptamer drugs.

[0043] In addition, the concept of the present invention can also be used to construct other protein nano double shell structures with α-helical endings similar to the target protein, so as to be applied to the structural research of other similar target proteins. In addition, this study uses the characteristics of natural protein self-assembly to form polymers to carry out protein reconstruction from a conformational perspective, which is popularizable and inspiring for protein research. Moreover, the interaction between the target protein and the ligand (the present invention is a nucleic acid aptamer) is maintained in this nanostructure, which is of great significance for the structural research of the protein-ligand interaction complex and the development of structure-guided targeted drugs.

[0044] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0045] The materials, reagents and instruments used in the embodiments of the present invention are as follows:

[0046] Materials: pMAL-c2X protein expression plasmid containing the target protein expression fragment (provided by General Biotechnology Co., Ltd., Chuzhou, Anhui), BL21 (DE3) super competent cells, 100 kD ultrafiltration tube, fluorescently labeled nucleic acid aptamer (5'-3' sequence: FAM-GGTGGGTGGGTTGGGTGG, Shanghai Bioengineering), nucleic acid aptamer without fluorescent label (5'-3' sequence: GGTGGGTGGGTTGGGTGG, Shanghai Bioengineering), Series S Sensor Chip CM5 chip (Cytiva), fluorescence polarization 96-well plate, transmission electron microscope grid support film 200 mesh (Zhongjing Keyi), 96-well crystallization plate.

[0047] Reagents: LB medium, IPTG inducer, ampicillin, Dulbecco's phosphate buffer, ethylenediaminetetraacetic acid (EDTA), imidazole, MBP purification filler (provided by Tiandirenhe Biotechnology Co., Ltd., Changzhou, Jiangsu), nickel column purification filler (provided by Tiandirenhe Biotechnology Co., Ltd., Changzhou, Jiangsu), TEV enzyme (Beyotime), SDS-PAGE, Tris-glycine electrophoresis buffer, Coomassie brilliant blue staining solution (Beyotime), 1.5% uranyl acetate (purchased from Zhongjing Keyi), Crystal Screen 2 TM Kit (Hampton Research), PEG / IonScreen Kit (Hampton Research).

[0048] Instruments: 37°C incubator and shaker, centrifuge, ultrasonic disruptor, Bio Rad vertical high-voltage electrophoresis system, EnVision2105 microplate reader, surface plasmon resonance molecular interaction system (Biacore-8K, SPR), isothermal titration microcalorimeter (Malvern Panalytical), GE protein purification system, ion chromatography column, BIO-RAD protein purifier, HiPrep16 / 60Sephacryl S-300High Resolution purification column (Cytiva), biological transmission electron microscope (JEM-2100plus), protein crystal incubator.

[0049] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0050] Example 1

[0051] This embodiment provides a method for constructing a protein nano double shell structure for electron microscopy and crystal research. The specific steps are as follows:

[0052] (1) Plasmid construction and transformation: The target protein was constructed in the pMAL-c2X protein expression vector. 1 μg of plasmid was added to 100 μL BL21 Escherichia coli competent cells and incubated on ice for 30 min. Heat shock was performed at 42°C for 60 s and ice bath was performed for 5 min. 500 μL LB (Luria-Bertani) medium without antibiotics was added and the culture was shaken at 37°C and 220 rpm for about 1 h. The plate was spread on an LB plate with an ampicillin concentration of 100 mg / L and incubated in an incubator at 37°C for 12-16 h. A single colony was picked for expansion.

[0053] Among them, the pMAL-c2X plasmid is an expression vector containing the nucleotide sequence of maltose binding protein. When constructing the expression vector, the amino acid sequence of the target protein includes a His tag sequence, a TEV hydrolase sequence, a C-terminal sequence of LRPPRC, and an Escherichia coli ferritin sequence. The specific amino acid sequence of the target protein is shown in SEQ ID NO: 3:

[0054] .

[0055] (2) Protein induction expression: When the bacterial solution expanded in step (1) reaches an OD600 of 0.6-1.0, add isopropyl β-D-Thiogalactoside (IPTG) inducer to a final concentration of 0.5 mM, induce overnight at 16°C, 220 rpm for about 16 h, and collect the bacteria by low-temperature centrifugation.

[0056] (3) Bacterial disruption: The cells were resuspended in DPBS buffer containing 5 mM imidazole, 0.05 mM EDTA, and 5% glycerol. After ultrasonic disruption, centrifugation was performed at 9800 rpm and 4°C for 1 hour, and the supernatant was collected.

[0057] (4) Protein purification and structural sample preparation:

[0058] 1) Affinity purification using MBP tag, washing with DPBS buffer, and eluting and collecting the protein with DPBS buffer containing 20 mM maltose.

[0059] 2) For electron microscopy imaging, the protein sample was run on polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue to confirm the correct band size. Then, the single-state protein was separated using HiPrep 16 / 60 Sephacryl S-300 High Resolution purification column in BIO-RAD protein purifier for negative staining electron microscopy imaging. The collected protein particle images were reconstructed in two dimensions using Resin software. Electron microscopy imaging and two-dimensional reconstruction: The obtained protein was concentrated to 1.0 mg / mL using an ultrafiltration tube, and 5 μL of protein was added to the hydrophilic copper mesh carbon film. After standing for 60 seconds, the filter paper was used to absorb the water. Then, 5 μL of 1.5% uranyl acetate was added to stain for 60 seconds, and the filter paper was used to absorb the water. After staining twice, the transmission electron microscope was used to image at 80KV voltage and 80k magnification, and the Resin software was used for two-dimensional reconstruction.

[0060] 3) For crystal screening, the protein collected by elution with DPBS buffer containing 20mM maltose in step 1) was eluted with TEV enzyme at 4°C overnight to remove the MBP tag, and purified with His tag. The DPBS flow-through containing 0mM imidazole, 10mM imidazole, 20mM imidazole, 30mM imidazole, and 300mM imidazole was collected respectively. After gel running confirmation, the protein without MBP tag was collected. The same as above step 2) was purified on the BIO-RAD protein purifier using HiPrep 16 / 60 Sephacryl S-300 High Resolution purification column to obtain a single state protein. Here, the LRPPRC-E.coli ferritin protein with the MBP tag removed was also imaged by electron microscopy and reconstructed in two dimensions using Resin software to confirm the successful assembly of the fusion protein, and then the hanging drop method was used with CrystalScreen 2 TM Crystal screening was performed using the PEG / Ion Screen kit and the PEG / Ion Screen kit. Protein crystal screening: The obtained protein was concentrated using an ultrafiltration tube and replaced with 100mM NaCl, 20mM Tris-HCl (pH8.0) buffer. The protein was spotted using a 96-well crystal plate. First, 40μL of commercial screening buffer was added to each large well, and then 1μL of protein was added to the small wells next to it. Then, 1μL of screening buffer was transferred from the large well to the corresponding small wells. After gently shaking until the protein and screening solution were mixed, the plate was immediately sealed with transparent tape and placed in a protein crystal incubator at room temperature for one week. The plate was then observed under a microscope to determine whether there were protein crystals.

[0061] Figure 3 The results of protein MBP affinity purification and electron microscopy characterization in step 2) are shown in Table 2. The size of the target protein plus the MBP purification tag is 102 kD. Figure 3A: Lanes 1, 2, and 3 are the protein supernatant washed with DPBS for the first, second, and third time after passing through the column, and lanes 4 and 5 are the target proteins eluted with DPBS containing 20 mM maltose; Figure 3 The protein obtained in A was further purified by molecular sieve purification (column model: HiPrep 16 / 60 Sephacryl S-300 High Resolution purification column (Cytiva)), as Figure 3 As shown in B, Figure 3 The corresponding peak in B was run on SDS-PAGE to confirm the bands and obtain Figure 3C. Lanes 1, 2, and 3 in Figure 3C are Figure 3 B A1, A2, A5 tubes for the first peak; Figure 3 Lanes 4, 5, and 6 of C are Figure 3 B The A11, A16, and A22 tubes of the second peak; Figure 3 Lanes 7, 8, and 9 of C are Figure 3 B The third peak of tubes A28, A32, and A35. Combined with the column separation instructions and protein gel strips, the second peak of the molecular sieve was determined to be the fusion protein monomer peak, and the protein concentrates in the corresponding tubes A11-A22 were collected; Figure 3 D is the electron microscopy imaging and 2D fitting results after protein negative staining. According to the final collected protein negative staining imaging morphology and the results of two-dimensional reconstruction using Resin software, it is shown that the fusion protein was successfully constructed, and the negative staining can preliminarily show the target protein morphology, which provides the possibility for further analysis of the structure using high-resolution cryo-electron microscopy.

[0062] Figure 4 The results of purification and crystal screening of the protein with the MBP tag cut off and the His tag reversed in step 3). Figure 4 A is a schematic diagram of cutting the MBP tag; Figure 4 B is the SDS-PAGE reverse hanging purification test, the marked swimming lane 1 is the product of the overnight reaction with TEV enzyme; the marked swimming lanes 2, 3, 5, 7, and 9 are the reaction products after the column flow through and the DPBS containing 0mM, 10mM, 20mM, 40mM, and 300mM imidazole is used to elute the collection solution, and the remaining swimming lanes are blank swimming lanes to prevent cross-sampling. The target protein in swimming lanes 3 and 5 is collected and passed through the molecular sieve, and the results are as follows Figure 4 C. Yes Figure 4 The corresponding peak in C was confirmed by SDS-PAGE. Figure 4 D, Figure 4 The lanes marked 1, 2, and 3 are Figure 4 C A2, A5, and A7 tubes for the first peak; Figure 4 The lanes marked 4, 5, and 6 are Figure 4 C A17, A22, and A30 tubes for the second peak; Figure 4 The lanes marked 7, 8, and 9 are Figure 4 C The third peak of A52, A56, and A64 tubes. Combined with the column separation instructions and protein gel strips, the second peak of the molecular sieve was determined to be the fusion protein monomer peak with the MBP tag cut off, and the protein concentrates in the corresponding tubes A17-A30 were collected. Figure 4 E is the electron microscopy imaging and 2D fitting results after negative staining of the protein, which also shows that the fusion protein with the tag removed is successfully constructed and the target protein spike exists. Then the sitting drop method is used to screen the crystals, such as Figure 4 F. Using Crystal Screen 2 TM Kit (Hampton Research), PEG / Ion Screen Kit (Hampton Research) was used to screen crystals using a 96-well crystallization plate, and Crystal Screen 2 was observed under a microscope. TM The reagents in kit No. 47 were screened to obtain protein crystals such as Figure 4 G. The final protein crystal conditions were room temperature incubation, Crystal Screen 2 TM 0.1M bicine (pH 9.0), 2.0M MgCl2 of the kit.

[0063] Example 2

[0064] The method for conducting electron microscopy and crystal research on the LRPPRC protein structure using the protein nano double shell structure obtained in Example 1 is as follows:

[0065] First, fluorescence polarization, surface plasmon resonance, and isothermal titration calorimetry were performed to verify the binding ability between the constructed fusion protein and the nucleic acid aptamer, including:

[0066] (A) Fluorescence polarization experiment: The assay buffer was DPBS buffer (Biosharp) containing 5 mM MgCl2. Each sample consisted of 100 μL of detection solution, which contained 50 nM FAM-labeled aptamers (50 μL) and different concentrations of LRPPRC fusion protein (50 μL). After mixing at room temperature, fluorescence polarization measurements were performed in the wells of a 96-well plate (Bioland Scientific). The experiment was performed using an EnVision2105 microplate reader (BioTek, USA). The excitation wavelength of FAM was set to 485 nm and the emission filter was set to 535 nm. The average Kd value of 3 replicates was determined by specific binding curve fitting using GraphPad Prism 9.0.0 software.

[0067] (B) Surface plasmon resonance experiment: Binding affinity was measured using the Biacore 8K SPR system at room temperature 25°C in DPBS buffer (Biosharp) with 5 mM MgCl2. 20 μg of LRPPRC protein was added to the activator 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride buffer (pH 3.6) and coupled to a CM5 chip (GE Healthcare): contact time 120 s, dissociation time 120 s, flow rate 30 μL / min. When the array concentration of aptamer R14 flew over the chip surface, the response units of aptamers at different concentrations were measured. Biacore Insight evaluation software used a 1:1 Langmuir binding model to analyze the binding kinetics and obtain the Kd value.

[0068] (C) Isothermal titration calorimetry experiments: Isothermal titration calorimetry experiments were performed on a PEAQ-ITC instrument (Malvern Panalytical). Aptamers and proteins were titrated in DPBS buffer (Biosharp) containing 5 mM MgCl2, and both proteins and aptamers were centrifuged at 13000g for 20 minutes before the experiment. 300 μL of LRPPRC protein was loaded into the cell chamber, and 70 μL of aptamer was loaded into the syringe. The titration experiment was performed at 25°C. The final thermodynamic values ​​were obtained by fitting the single-site binding model using Origin software.

[0069] Figure 5 The results of the interaction between the constructed fusion protein containing the target protein and the nucleic acid aptamer are verified. A, B, and C are the results of fluorescence polarization, surface plasmon resonance, and isothermal titration calorimetry, respectively, proving that the target protein can still have a good affinity with the nucleic acid aptamer after recombinant.

[0070] Experimental Example 3

[0071] This experiment compares the purification effects of different affinity purification methods, including: His tag purification (nickel column purification filler, provided by Tiandirenhe Biotechnology Co., Ltd., Changzhou, Jiangsu), strong anion exchange chromatography Capto Q column purification (HiTrapCapto Q chromatography column, Cytiva; GE protein purification system, avant 25 model), strong cation exchange Capto S column purification (HiTrap Capto S chromatography column, Cytiva; GE protein purification system, avant 25 model).

[0072] Figure 6 The results of affinity purification using other methods are shown in Table 1. Figure 6A is the result of SDS-Page detection of His-tag nickel column purification. The marked channel 1 is the bacterial solution, channel 2 is the protein flow-through collection solution, channels 3, 4, and 5 are DPBS washing collection solutions containing 10mM, 20mM, and 40mM imidazole, respectively, and channel 6 is DPBS elution collection solution containing 300mM imidazole, which is the target protein with a size of 102kD. Figure 6 A indicates that the purity of His tag purification is not as good as that of MBP purification tag. Ion exchange column is also a common method for further purification of proteins, so GE protein purification system is also used to try to purify nanostructures. The isoelectric point of most proteins in nature is around 5.8 or 9, and most of them are acidic. For unknown samples, strong anion exchange chromatography Capto Q column is generally used for purification, using Tris neutral buffer at pH 7.5. Figure 6 As shown in B; the Q column was generally sticky, so a strong cation exchange Capto S column was used for purification, using a pH 5.5 Tris buffer. Figure 6 C. Figure 6 B and Figure 6 Table C is the statistics of protein purification peaks automatically generated by the instrument. Figure 6 D is the SDS-PAGE gel run confirmation result of the protein purification peaks of the Q column and S column. The column flow-through FT was used as a control, and the A9, B8, C1, C5, and D1 tubes of the Q column, and the A9, A12, and B6 tubes of the S column were taken for gel run confirmation. Figure 6 B. Figure 6 C. Figure 6 D Results show that both the Q column and the S column of this nanostructure have some hanging column, and the purification purity is acceptable, but the binding effect of both the Q column and the S column is not particularly good, and most of the protein becomes the flow-through liquid. It can be seen that ion affinity chromatography is not a very suitable purification method for the nanostructure of MBP-LRPPRC-ferritin. In particular, Figure 6 E is for Figure 6 D shows the TEM photography result of the corresponding protein with good purity collected after concentration and negative staining. Although it seems to have a certain nano-morphology, its contrast granularity is not strong in the same negative staining imaging operation, which is far worse than the TEM photography effect obtained after molecular sieve purification. This shows that the second single peak obtained by MBP tag purification and molecular sieve further purification is a better choice for structural research, whether TEM imaging morphology or crystal screening and growth, compared with ion affinity chromatography.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A protein nano double shell structure for electron microscopy and crystal research, characterized in that: The protein nano double shell structure comprises an inner shell, an outer shell and a target protein sandwiched between the inner and outer shells. The inner shell is ferritin, and the ferritin comprises ferritin derived from Escherichia coli.

2. The protein nano double shell structure according to claim 1, characterized in that: The target protein is a protein segment ending in an alpha helix; The target protein is LRPPRC protein, and the protein segment can interact with the nucleic acid aptamer.

3. The protein nano double shell structure according to claim 2, characterized in that: The shell is a tag protein; Preferably, the tag protein is maltose binding protein.

4. The method for constructing a protein nano double shell structure according to any one of claims 1 to 3, characterized in that: include: The expression vectors in which the genes of the tag protein, the target protein and the ferritin are connected in sequence are introduced into the host bacteria, the protein expression is cultured and induced, and the protein nano double shell structure can be obtained after separation and purification.

5. The construction method according to claim 4, characterized in that: The amino acid sequence of the target protein is shown in SEQ ID NO: 1, and the amino acid sequence of the ferritin is shown in SEQ ID NO:

2.

6. The construction method according to claim 4, characterized in that: The purification includes: protein tag purification and molecular sieve treatment; Preferably, the tags used in the protein tag purification process include: MBP tag and / or His tag; Preferably, the maltose binding protein-LRPPRC-ferritin derived from Escherichia coli is obtained by purification using the MBP tag; Preferably, LRPPRC-E. coli-derived ferritin is obtained by the MBP tag purification and His tag reverse hanging.

7. The use of the protein nano double shell structure according to any one of claims 1 to 3 in electron microscopy and crystal research of target protein, characterized in that: include: The purified protein nano double-shell structure is subjected to two-dimensional reconstruction by transmission electron microscopy imaging to determine the structure of the target protein in the protein nano double-shell structure; and / or The purified protein nano double shell structure is cut off from the MBP tag, and the protein crystals expressing the target protein fusion are obtained through filtration, concentration and hanging drop screening.

8. A fusion protein, characterized in that The method comprises: ferritin derived from E. coli, a target protein expressed by fusion with the N-terminus of ferritin derived from E. coli, and a tag protein connected to the N-terminus of the target protein; The target protein is LRPPRC protein; Preferably, the tag protein is maltose binding protein.

9. A carrier, characterized in that Comprising an expression cassette for expressing the fusion protein according to claim 8.

10. A kit, characterized in that: It comprises the protein nano double shell according to any one of claims 1 to 3, the fusion protein according to claim 8 or the vector according to claim 9.

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