Protein structure and analytical method thereof

Abstract

The invention relates to the technical field of biology, and relates to a protein structure and an analytical method thereof, in particular to a tobacco NtTCTP protein structure and an analytical method thereof. The invention verifies that NtTCTP exists in the form of a dimer, the potential interaction positions of two proteins are amino acids of a beta-folded sheet part, the measurement result of the molecular spacing is about 3 angstroms, and the range of the amino acids participating in interaction is Q65 to V70.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and relates to a protein structure and an analysis method thereof, and in particular to a TCTP protein structure and an analysis method thereof. Background Art

[0002] Potato virus Y (genus Potatovirus, family Potyviridae) is the largest class of RNA viruses known to infect plants and includes many agriculturally important viruses that cause devastating epidemics and yield losses in many crops around the world. Several potato virus Y species are listed as the top ten most scientifically important plant viruses. Potato virus Y can cause wilting, mosaic, vein necrosis, and sometimes stunting and necrosis in tobacco, tomato, and other economically important plants. Potato virus Y is a class of disaster viruses that can cause tobacco yield reduction and seriously affect tobacco leaf quality. Potato virus Y has been studied more than other plant viruses, and the crystal structure of the NtTCTP protein has important research and application value.

[0003] The phase problem is the core problem in the process of protein crystallographic structure analysis. The general methods for analyzing the diffraction phase of protein crystals are: molecular replacement method (i.e. phase simulation), isomorphous replacement method and anomalous scattering method. If there are similar homologous structures in the protein data that have been analyzed at this stage, the phase can be analyzed by molecular replacement method. However, the results of molecular replacement of NtTCTP protein are not good. The software cannot completely solve the structure of TCTP, and a large part of the structure is missing. If a new structure is to be analyzed, it is necessary to rely on isomorphous replacement method or anomalous scattering method. In crystallography, mad (multi-wavelength anomalous dispersion) is often used to infer the phase. Compared with sulfur atoms, the anomalous scattering signal of heavy metal atoms selenium is clearer, so selenomethionine-labeled proteins are increasingly used in mad analysis phase experiments. The chemical properties of selenium are very similar to those of sulfur; secondly, selenomethionine can completely replace methionine without changing the properties of the replaced protein; thirdly, most protein crystals with selenomethionine replacing methionine are isomorphous crystals with the corresponding natural protein crystals, which are more reliable.

[0004] The principle of protein crystal structure analysis is mainly based on X-ray diffraction technology. When X-rays irradiate a crystal, the atoms in the crystal will scatter the X-rays, and the intensity and direction of these scattered lights are related to the crystal structure. By measuring the intensity and direction of these scattered lights, the positions and arrangements of atoms in the crystal can be determined, thereby obtaining the three-dimensional structure information of the protein. The electron density map is the key in X-ray crystallography. It visually shows the positions where electrons accumulate, thus indicating the possible positions of atoms in the molecule. The regions where electrons accumulate may contain one or more atoms, and these regions appear as high-density areas on the electron density map, which is the basis for protein crystal structure analysis. In this process, the electron density map provides direct evidence of the atomic positions. In a protein crystal, high electron density regions usually correspond to the atomic positions in the protein molecule, while low electron density regions may correspond to solvent molecules or other non-protein molecules. The distribution and characteristics of these density regions are crucial for understanding the structure and function of the protein. Summary of the Invention

[0005] In view of the above problems, the present invention provides a TCTP protein structure and its analysis method. To achieve this purpose, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides an analysis method for NtTCTP protein, which is characterized in that selenomethionine is added to the culture medium.

[0007] In the present invention, the inventors found that the method of culturing with selenomethionine is to artificially add selenomethionine to the culture medium, so that methionine in the synthesized protein is replaced by selenoamino acid. When crystal diffraction occurs, the software will recognize the signal of heavy metal selenium. During the distribution of the density region, this region is directly recognized as selenomethionine, and then other density regions will use this as a reference. In this way, the phase analyzed is the true phase.

[0008] According to the present invention, the concentration of selenomethionine is 30 - 100 mg / L, specifically including 30 mg / L, 32 mg / L, 34 mg / L, 35 mg / L, 36 mg / L, 38 mg / L, 40 mg / L, 42 mg / L, 44 mg / L, 45 mg / L, 46 mg / L, 48 mg / L, 50 mg / L, 52 mg / L, 54 mg / L, 55 mg / L, 56 mg / L, 58 mg / L, 60 mg / L, 62 mg / L, 64 mg / L, 65 mg / L, 66 mg / L, 68 mg / L, 70 mg / L, 72 mg / L, 74 mg / L, 75 mg / L, 76 mg / L, 78 mg / L, 80 mg / L, preferably 50 - 80 mg / L, and further preferably 60 mg / L.

[0009] According to the present invention, the method comprises the following steps:

[0010] Prokaryotically express the NtTCTP fusion protein, purify the protein, and analyze the protein structure by X-ray diffraction. Among them, selenomethionine is added during the cultivation of the cloning bacteria for prokaryotic expression of the NtTCTP fusion protein.

[0011] In the present invention, the concentration of selenomethionine is 30 - 100 mg / L, preferably 50 - 80 mg / L, and further preferably 60 mg / L.

[0012] In a second aspect, the NtTCTP protein structure obtained by analyzing according to the analysis method described in the first aspect.

[0013] In a third aspect, the analysis method as described in the first aspect is used to analyze the structure of a protein.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention verifies that NtTCTP exists in the form of a dimer. The potential interaction positions of the two proteins are the amino acids in the β-sheet part. The measurement result of the molecular spacing is about 3 Å, and the range of amino acids participating in the interaction is from Q65 to V70. Description of the Drawings

[0016] Figure 1 For the purification of NtTCTP expressed by the strain, where 1: total protein after expression; 2: flow-through after expression; 3 - 6: samples harvested after 1 - 4 times of elution; 7: Marker;

[0017] Figure 2 For the result after purification of NtTCTP, where M: Marker, S: sample of NtTCTP after purification.

[0018] Figure 3 Amino acids on the β-sheet of the present invention with relatively close distances; Detailed Embodiments

[0019] To further elaborate on the technical means and effects adopted by the present invention, the following further illustrates the technical solution of the present invention in conjunction with the drawings and through specific embodiments, but the present invention is not limited to the scope of the embodiments.

[0020] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular commercial channels.

[0021] Example 1: Experimental Steps for Prokaryotic Expression of Se-MetNtTCTP Fusion Protein

[0022] (1) Transfer the correctly sequenced pET-13-NtTCTP plasmid into Rosetta competent cells. Use a sterilized pipette tip to pick a single colony and inoculate it into an LB liquid medium with Amp resistance. Incubate overnight at 37°C and 220 rpm in a shaker.

[0023] (2) The next day, transfer the turbid bacterial solution to a selenide adaptation medium at a ratio of 1:100. Incubate at 37°C and 220 rpm in a shaker for 6 - 8 hours.

[0024] (3) Collect the bacterial solution using a centrifuge bottle. Centrifuge at 4000 rpm for 10 min to remove the supernatant. Resuspend the cell pellet with the expression medium and finally transfer it to a new expression medium. Incubate at 37°C and 220 rpm in a shaker until the OD 600 reaches 0.6, then adjust the shaker temperature to 16°C.

[0025] (4) Use 0.5 M Na 3 PO 4 to adjust the pH value of the expression medium to 7.2. Then weigh the amino acids proportionally: 100 mg / L of lysine, threonine, and phenylalanine, 50 mg / L of leucine, isoleucine, and valine, and 60 mg / L of selenomethionine. Store them in a light-protected centrifuge tube.

[0026] (5) Add the amino acids to the expression medium. Incubate at 16°C and 220 rpm for 1 h, then add IPTG to a final concentration of 0.5 mM / L and continue the incubation.

[0027]

[0028] Store at room temperature for later use.

[0029]

[0030] Sterilize at 121°C for 20 min.

[0031]

[0032] Filter sterilize with a 0.22 μm filter membrane and store at 4°C in a refrigerator for later use.

[0033]

[0034] Filter sterilize with a 0.22 μm filter membrane and store at 4°C in a refrigerator for later use.

[0035]

[0036] Example 2: Purification of NtTCTP Fusion Protein

[0037] The study of the NtTCTP protein structure requires highly pure protein, and the purity of the target NtTCTP protein is improved through multiple-step purification. The purification process of the NtTCTP protein in the present invention is as follows: Escherichia coli is lysed by high-pressure crushing to release the proteins in the cells; centrifugation is used for preliminary solid-phase separation; a variety of purification methods such as nickel column affinity chromatography, anion exchange chromatography, and gel filtration chromatography are combined to obtain a protein with good homogeneity and high purity for subsequent crystallization screening experiments. Specifically as follows:

[0038] After optimizing the expression conditions, host strains, and purification strategies, the following expression and purification scheme was confirmed: Using Rossetta as the expression strain, the results of the preliminary purification elution are as Figure 1 shown in lanes 3-6 in the figure: As the number of elution times increases, the amount of the product decreases, but the purity of the product increases. Considering the feeding ratio and the experimental purpose comprehensively, the sample eluted once is used for the subsequent purification steps.

[0039] In the subsequent purification, after collecting the first elution sample after Ni-NTA affinity chromatography, chromatography is carried out using Superdex75 prep grade. After gel chromatography, ultrafiltration is performed, and the obtained sample is detected by WB using His antibody. The results are as Figure 2 shown: After ultrafiltration, there is an obvious large amount of expression signal at the position of the target protein with a size of about 30-40 kDa, and the signal is relatively single, indicating that the expression and purification of pET28b-NtTCTP are successful and can be used for subsequent interaction experiments.

[0040] Example 3: Diffraction analysis of the NtTCTP fusion protein

[0041] The structure of NtTCTP is analyzed by X-ray diffraction technology. By measuring the intensity and direction of these scattered lights, the positions and arrangements of atoms in the crystal can be determined, thereby obtaining the three-dimensional structure information of the protein, which usually appears as an electron density map. Through computer programs for data processing, analysis, and model establishment, by multiplying the intensity and direction data of the scattered lights, combining crystallographic principles and mathematical calculation methods, the space group of the unit cell, the unit cell parameters, and the coordinate positions of atoms can be inferred, thereby establishing a three-dimensional structure model of the protein, specifically as Figure 3 shown.

[0042] The method of culturing with selenomethionine is to artificially add selenomethionine to the culture medium, so that methionine in the synthesized protein is replaced by seleno-amino acids. When the crystal diffracts, the software will recognize the signal of heavy metal selenium. During the distribution in the density region, this region is directly recognized as selenomethionine, and then other density regions will use this as a reference. In this way, the phase resolved is the real phase. In the structural analysis of NtTCTP, we found that the two α-helices in the protein structure are quite different from the TCTP protein structures of other published species.

[0043] Further analysis through the present invention reveals that NtTCTP exists in the form of a dimer. The potential interaction positions of the two proteins are the amino acids in the β-sheet part, the measurement result of the molecular spacing is about 3 Å, and the range of amino acids participating in the interaction is from Q65 to V70.

[0044] The applicant declares that the present invention uses the above embodiments to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for analyzing NtTCTP protein, characterized in that: The method comprises adding selenomethionine to a culture medium for culturing clonal bacteria.

2. The analytical method according to claim 1, characterized in that: The concentration of selenomethionine is 30-100 mg / L.

3. The analysis method according to claim 2, characterized in that: The concentration of selenomethionine is 50-80 mg / L, preferably 60 mg / L.

4. The analytical method according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: The NtTCTP fusion protein is expressed in prokaryotes, the protein is purified, and the protein structure is analyzed by X-ray diffraction, wherein selenomethionine is added during the culture of the cloned bacteria expressing the NtTCTP fusion protein in prokaryotes.

5. The analysis method according to claim 4, characterized in that: The concentration of selenomethionine is 30-100 mg / L.

6. The analysis method according to claim 5, characterized in that: The concentration of selenomethionine is 50-80 mg / L, preferably 60 mg / L.

7. The NtTCTP protein structure obtained by analyzing the method according to any one of claims 1 to 6.

8. The analytical method according to any one of claims 1 to 6 is used for analyzing the structure of a protein.

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