Preparation and crystallization method of castor Cdc5-DBD protein
Through prokaryotic expression system and multi-layer purification technology, high-quality Cdc5-DBD protein crystals were obtained, which solved the difficulties in protein expression and crystallization in the prior art and met the needs of structural biology research.
Patent Information
- Application Number
- CN202411628558.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-14
- Publication Date
- 2025-01-03
AI Technical Summary
Since the structure of the Cdc5-DBD protein is still unclear, the binding mechanism with DNA needs to be further studied. It is difficult to obtain high-quality Cdc5-DBD protein samples and high-quality crystals with good stability in the prior art, which limits the progress of structural biological analysis. .
The fusion expression vector was constructed using a prokaryotic expression system, and the purified Cdc5-DBD protein was purified by IPTG induction expression and combined with affinity chromatography and molecular sieve chromatography, and crystallized under specific buffer conditions to obtain high-quality single crystals.
The Cdc5-DBD protein crystals with high purity, large expression and high resolution are achieved, which meets the research needs of protein-DNA binding interaction mechanism and provides better operability and repetition.
Smart Images
Figure CN119242672A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of protein engineering, and in particular to a method for preparing and crystallizing castor Cdc5-DBD protein. Background Art
[0002] As an important energy and oil crop, castor is resistant to drought, saline-alkali and barrenness. It is a dominant crop on saline-alkali land and can be used to improve the ecology of saline-alkali land and increase farmers' income in saline-alkali areas.
[0003] The Prp19 complex affects the ricinoleic acid synthesis pathway. The core skeleton protein of the Prp19 complex, Cdc5, and the N-terminal DNA-Binding Domain (DBD) sequence are involved in DNA binding. During the DNA repair process, it guides the repair Prp19 complex to quickly locate to the DNA damage site and control the cell cycle checkpoint, causing the damaged cell cycle to arrest. Cdc5-DBD is highly conserved throughout the eukaryotic system.
[0004] Since the structure of Cdc5-DBD protein is still unclear and its binding mechanism with DNA needs further study, obtaining high-quality Cdc5-DBD protein samples and stable, reproducible high-quality crystals is of great significance for advancing structural biology to analyze the binding interaction mechanism of Cdc5-DBD with DNA. Summary of the invention
[0005] The purpose of the invention is to provide a method for preparing and crystallizing castor Cdc5-DBD protein.
[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0007] The present invention provides a method for preparing castor Cdc5-DBD protein, comprising the following steps:
[0008] (1) Construction of fusion expression vector;
[0009] The target gene of Ricinus communis Cdc5-DBD protein was cloned into the expression vector to construct a fusion expression vector;
[0010] (2) Expression of target gene
[0011] The fusion expression vector is transferred into the competent state, and the expression is induced by IPTG; the bacterial solution induced by IPTG is collected, and the bacterial cells are collected after the bacterial solution is centrifuged;
[0012] (3) Purification of target protein by affinity chromatography
[0013] After the cells are resuspended, they are mixed with protease inhibitors and reducing agents, and the mixed solution is ultrasonically disrupted and centrifuged in sequence to prepare the target protein supernatant; after the Ni-affinity chromatography column is equilibrated with affinity buffer, the target protein supernatant is gradient eluted;
[0014] (4) Purification of target protein by molecular sieve chromatography
[0015] After the molecular sieve chromatography column was equilibrated with molecular sieve chromatography buffer, the 400 mM imidazole eluate was collected and the target protein was further purified according to the protein size by molecular sieve chromatography.
[0016] Preferably, the nucleotide sequence of the target gene of the Ricinus communis Cdc5-DBD protein in step (1) is as shown in SEQ ID NO.5:
[0017] atgaggataatgataaaaggaggagtgtggaagaacacggaggatgagatactgaaagcggcggttatgaaatatggaaaaaatcaatgggctcgtatctcttctcttcttgttcgtaagtctgctaaacaatgtaaggctcgctggtatgagtggcttgacccc tccattaaaaagactgagtggaccagagaggaggatgagaaattgcttcatcttgctaagctcatgccaactcaatggcgaactattgctccaattgttggccgtactccgtctcagtgtctcgagcgatatgagaagcttcttgatgctgcgtgtgttaaggat;
[0018] Its amino acid sequence is shown in SEQ ID NO.6:
[0019] MRIMIKGGVWKNTEDEILKAAVMKYGKNQWARISSLLVRKSAKQC KARWYEWLDPSIKKTEWTREEDEKLLHLAKLMPTQWRTIAPIVGRTPSQ CLERYEKLLDAACVKD.
[0020] Preferably, the induced expression in step (2) is induced by culturing with 0.8 mM IPTG, 18° C., and 160 rpm for 12 h.
[0021] Preferably, in step (3), the protease inhibitor is PMSF and the reducing agent is DTT.
[0022] Preferably, the affinity chromatography buffer in step (3) has a formula of 20 mM MES, 150 mM NaCl, 1 mM DTT, pH 6.0.
[0023] Preferably, the formula of the molecular sieve chromatography buffer in step (4) is 20mM MES, 150mM NaCl, pH 6.0.
[0024] The present invention also provides a crystallization method of castor Cdc5-DBD protein. The castor Cdc5-DBD protein prepared by the preparation method of castor Cdc5-DBD protein is prepared into a protein solution with a concentration of 5 mg / mL using a buffer solution, and then the solution is statically cultured in a 16° C. constant temperature incubator for 7 days in the presence of an MPD precipitant to obtain a single crystal of castor Cdc5-DBD protein.
[0025] Preferably, the MPD precipitant is an MPD aqueous solution, wherein the volume concentration of the MPD aqueous solution is 10-30%.
[0026] Preferably, the formula of the buffer is 20 mM MES, 150 mM NaCl, pH 6.0.
[0027] Compared with the prior art, the present invention has the following beneficial effects:
[0028] The present invention uses a prokaryotic expression system to express castor Cdc5-DBD protein, and after bacterial lysis, the lysis supernatant is sequentially passed through an affinity chromatography column and a molecular sieve chromatography column to finally obtain a purified target protein. In an MES buffer environment, the expression and purification of castor Cdc5-DBD protein is easier to operate, the protein expression amount is larger, the purity is higher, and the protein properties are better. The present invention also provides a crystallization method for castor Cdc5-DBD protein. Under 25% MPD conditions, a large number of single crystals of castor Cdc5-DBD protein can be obtained, and the obtained crystals are large, of good quality, and of high resolution, and the method has good repeatability; the crystals obtained by the crystallization method of the present invention have high resolution and can better meet the needs of research on the binding interaction mechanism of proteins and DNA. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 The SDS-PAGE electrophoresis diagram for detecting the protein induction expression effect in Example 1; wherein, lane 1 is a control, and lane 18°C indicates induction at a temperature of 18°C;
[0030] Figure 2The SDS-PAGE electrophoresis diagram of the protein samples in each slurry during the affinity chromatography process in Example 1; wherein, S is Supernatant, which is the total protein in the supernatant after centrifugation; P is Precipitate, which is the total protein in the precipitate after centrifugation; FT is Flow through, which is the total protein that flows through; 0-400 respectively indicates that the eluent (20mM MES, pH 6.0, 150mM NaCl) contains 0mM, 20mM, 80mM, and 400mM imidazole; M is a protein standard marker;
[0031] Figure 3 The molecular sieve chromatogram (SuperdexTM 75 10 / 300GL) of the protein sample in Example 1 and the SDS-PAGE electrophoresis diagram of the main peak of the molecular sieve of the protein sample;
[0032] Figure 4 The crystal images of the pETM10-Cdc5-DBD protein in Example 2; wherein Figure A is 20% MPD and Figure B is 25% MPD.
[0033] Figure 5 This is the diffraction pattern of the pETM10-Cdc5-DBD protein crystal in Example 2. DETAILED DESCRIPTION
[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0035] Example 1 Preparation method of castor Cdc5-DBD protein
[0036] 1. Construction of expression vector
[0037] (1) Primer design
[0038] The 1-110AA sequence of the castor Cdc5 (XP_002521973.1) gene was searched in NCBI, and the primers required for the sticky-end method were designed using Snap Gene. The specific primer sequences used in the present invention are shown in Table 1.
[0039] Table 1 Specific primer sequences
[0040]
[0041] (2) PCR amplification and recovery of target genes
[0042] During the PCR amplification of the target gene, the denaturation temperature of the reaction system is adjusted according to the activity of the PCR polymerase, the annealing temperature is adjusted accordingly according to the primer annealing temperature, and the extension time is determined according to the activity of the PCR polymerase and the fragment length of the target gene. The specific PCR amplification system and reaction parameters are shown in Table 2 and Table 3, respectively.
[0043] Table 2 PCR amplification system
[0044]
[0045] Table 3 PCR reaction parameters
[0046]
[0047]
[0048] The PCR product was subjected to 1% agarose gel electrophoresis to detect the size and purity of the target fragment (with reference to DNA Marker). The target fragment was recovered by gel excision, and the concentration of the recovered product was detected by Nano Drop micro-spectrophotometer for later use.
[0049] (3) DNA renaturation produces sticky ends
[0050] The purified products of F1 / R1 and F2 / R2 were mixed at a molar ratio of 1:1 and added with 1× Pryrobest PCR Buffer. The renaturation parameters were: 94°C for 5 min, 65°C for 15 min, and cooling at 16°C.
[0051] (4) Enzyme digestion of expression vector
[0052] In the present invention, two endonucleases, Nco I and Xho I, are mainly used to treat the expression vector, and the double enzyme digestion is carried out at 37° C. for 5-6 hours. The enzyme digestion system is shown in Table 4.
[0053] Table 4 Enzyme digestion system
[0054]
[0055] The digestion products were detected by 1% agarose gel electrophoresis, and the target fragments were recovered by gel excision (refer to DNA fragment rapid recovery kit).
[0056] (5) Connecting the vector to the target fragment
[0057] According to the T4 DNA Ligase instruction manual, the denatured product with sticky ends was mixed with plasmid pETM10 and incubated overnight at 16°C. The ligation system is shown in Table 5.
[0058] Table 5 Sticky end connection system
[0059]
[0060]
[0061] (6) Heat shock transformation and resistance screening
[0062] Competent cells E.coli DH5α were thawed on ice;
[0063] Add the ligation product to 50 μL competent cells and incubate on ice for 30 min;
[0064] Incubate in 42°C water bath for 90 seconds, then immediately cool on ice for 45 seconds;
[0065] Aseptically add 500 μL SOC medium and resuscitate at 37°C and 220 rpm for 30 min;
[0066] Spread the cultured bacteria evenly on an LB plate containing the resistance corresponding to the vector;
[0067] Invert and culture in a 37°C constant temperature incubator overnight, pick out single clone colonies and send to the company for sequencing.
[0068] 2. Expression of target protein
[0069] (1) Heat-shock transform the correctly sequenced plasmid into the E. coli expression strain E. coli BL21 (DE3);
[0070] (2) Pick a single clone and inoculate it into LB liquid medium containing resistance, culture it at 37°C and 220 rpm for 5-6 h, and the OD value is about 0.6-0.8;
[0071] (3) Transfer to 750 mL LB resistance medium;
[0072] (4) Cultivate at 37°C and 220 rpm for 4-6 h until the OD600 value reaches 0.6-0.8;
[0073] (5) Add 1 mM IPTG and induce culture at 18°C and 180 rpm for 15 h;
[0074] (6) Centrifuge at 12,800 × g for 10 min at room temperature, discard the supernatant, and collect the cells.
[0075] SDS-PAGE electrophoresis to detect protein expression (see Figure 1 ). Under the conditions of 1mM IPTG, 18°C, and 180rpm induction culture for 15h, the protein was expressed significantly, meeting the protein purification conditions.
[0076] 3. Purification of target protein
[0077] (1) Affinity chromatography
[0078] In prokaryotic expression, the simplest and most effective protein purification method is to bind metal ions with a 6×His tag to the recombinant protein. The metal ion (nickel ion or zinc ion) affinity chromatography used in the present invention is a method of extracting the 6×His recombinant protein by fixing the metal ions.
[0079] The theoretical molecular weight of Cdc5-DBD is MW = 12.3 kDa, and the isoelectric point is 9.2. Use 20 mM MES, pH 6.0, 150 mM NaCl, and 1 mM DTT as the affinity buffer. Resuspend the bacteria in 80 mL of affinity buffer, stir or vortex to disperse the bacteria and make the bacterial solution uniform;
[0080] Add protease inhibitor PMSF (final concentration 1 mM) and reducing agent DTT (final concentration 1 mM), stir to mix, and operate on ice;
[0081] Ultrasonic cell disruption for 10 min (2sec on / 4sec off, power 400W), ice bath operation; the ideal effect after ultrasonic disruption is that the cells are transparent like egg white;
[0082] The broken bacterial slurry was divided into 50 mL centrifuge tubes and centrifuged at 12 800 × g, 4 °C for 20 min.
[0083] Balance: Use 10 mL (5-10 column volumes) of affinity buffer to balance the His beads;
[0084] Sample loading: Add the protein supernatant obtained by centrifugation to His beads. Under the action of gravity, the slurry flows through the His beads. To ensure that the protein is fully chelated, repeat the sample loading twice;
[0085] Washing: Use 20 mL of affinity buffer containing 20 mM imidazole to wash His beads to remove non-specifically bound impurities and 80 mM imidazole to remove weakly bound recombinant proteins;
[0086] Elution: Use high concentration imidazole (400mM) affinity buffer to elute the target protein;
[0087] Electrophoresis: SDS-PAGE electrophoresis is used to detect the protein samples in each step of the slurry;
[0088] Filler storage: Wash His beads with 50mM EDTA to remove metal ions, then wash with ddH2O several times, seal the column with 20% ethanol, and store at 4℃.
[0089] After the bacteria were ultrasonically disrupted, they flowed through the Ni-sepharose column under gravity, and non-specific binding proteins were removed by 80mM low-concentration imidazole buffer, and the target protein was competitively eluted by 400mM high-concentration imidazole buffer. Figure 2 ) analysis, castor Cdc5-DBD protein has a high solubility in MES pH 6.0 Buffer environment. In the process of removing impurities with 80mM low concentration imidazole buffer, the target protein basically did not dissociate from the affinity chromatography filler column. Under the competition of high concentration imidazole (400mM), the target protein was eluted with less impurity proteins, achieving the purpose of affinity chromatography removal.
[0090] (2) Molecular sieve chromatography
[0091] The relatively pure protein sample obtained by affinity chromatography was further purified by molecular sieve chromatography based on protein size and aggregation state. The molecular sieve chromatography buffer used was 20 mM MES, pH 6.0, 150 mM NaCl.
[0092] Equilibration: Use 2 column volumes of buffer to wash the gel filtration chromatography column sample onto the Surperdex 7510 / 300GL gel column;
[0093] Sample loading: 0.5 mL of sample is automatically and slowly flowed into the chromatography column through the protein purification system;
[0094] Elution: Driven by the buffer, the protein sample flows through the molecular sieve chromatography column in sequence according to the molecular size, achieving the purpose of separation and purification;
[0095] Detection: The protein purifier detects the A280 nm absorption peak of the protein sample to determine the protein size and aggregation state.
[0096] The target protein purified by affinity chromatography was collected and further purified by molecular sieve chromatography according to the protein size. The main peak of pETM10-Cdc5-DBD protein was detected at 13.4 mL (see Figure 3 ), according to the standard, the protein size is about 11kDa, which is close to the theoretical molecular weight of 12.3kDa, indicating that the pETM10-Cdc5-DBD protein exists as a monomer in MES pH 6.0 Buffer. The pETM10-Cdc5-DBD main peak protein was collected, concentrated to 8mg / mL, dispensed into EP tubes, frozen in liquid nitrogen, and stored at -80℃ for later use.
[0097] Example 2
[0098] Crystallization Method of Ricinus communis Cdc5-DBD Protein
[0099] 1. Preparation of Castor Cdc5-DBD Protein Crystals
[0100] The pETM10-Cdc5-DBD protein prepared in Example 1 was dissolved in a buffer solution (20 mM MES, pH 6.0, 150 mM NaCl) to obtain a protein solution with a concentration of 5 mg / mL. Then, the shotgun crystal kit screening was performed. After standing for one week in a 16°C constant temperature incubator, the pETM10-Cdc5-DBD protein crystallized in a pool solution containing 20% MPD ((v / v)%) precipitant. The crystals were hexagonal flakes with stacking (see Figure 4 A). In order to obtain single crystals with better crystal form and suitable for diffraction, the crystallization conditions need to be further optimized.
[0101] Based on the original 20% MPD (v / v)% precipitant condition, 10%, 15%, 18.5%, 20%, 22.5%, 25%, and 30% were used for gradient screening of crystal growth conditions. The results showed that under the 25% MPD precipitant condition, hexagonal rhombus single crystals suitable for diffraction were observed (see Figure 4 B).
[0102] 2. Crystal diffraction and data collection
[0103] Preparation of antifreeze solution: add 20% glycerol to the crystal growth pool solution and mix well;
[0104] Crystal freezing: operate under a microscope, select a loop that is slightly larger than the crystal, and transfer the crystal from the original slurry to the antifreeze solution under the action of surface tension. The crystal adheres to the loop and is quickly transferred to liquid nitrogen for freezing;
[0105] Crystal diffraction: Crystal diffraction was performed at the Shanghai Synchrotron Radiation Source.
[0106] The crystals were transferred to an antifreeze solution containing 20% glycerol using a loop, quickly frozen in liquid nitrogen, and sent to the Shanghai Synchrotron Radiation Center for X-ray diffraction (see Figure 5 ). Crystal diffraction wavelength The distance from the detector is 300 mm, the exposure time is 0.5 s, and the crystal resolution is Each diffraction pattern was tilted at 1°, and a total of 360° was collected. Diffraction data analysis showed that it belongs to the C 2 2 21 space group, and the unit cell parameters α=β=γ=90°. See Table 6 for specific collection parameters.
[0107] Table 6 pETM10-Cdc5-DBD protein crystal structure data collection and processing
[0108]
[0109]
[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for preparing castor Cdc5-DBD protein, characterized in that: The following steps are involved: (1) Construction of fusion expression vector; The target gene of Ricinus communis Cdc5-DBD protein was cloned into the expression vector to construct a fusion expression vector; (2) Expression of target gene The fusion expression vector is transferred into the competent state, and the expression is induced by IPTG; the bacterial solution induced by IPTG is collected, and the bacterial cells are collected after the bacterial solution is centrifuged; (3) Purification of target protein by affinity chromatography After the bacterial cells are resuspended, they are mixed with protease inhibitors and reducing agents, and the mixed solution is ultrasonically disrupted and centrifuged to prepare the target protein supernatant; after the Ni-affinity chromatography column is equilibrated with affinity buffer, the target protein supernatant is gradient eluted; (4) Purification of target protein by molecular sieve chromatography After the molecular sieve chromatography column was equilibrated with molecular sieve chromatography buffer, the 400 mM imidazole eluate was collected and the target protein was further purified according to the protein size by molecular sieve chromatography.
2. The preparation method according to claim 1, characterized in that: The nucleotide sequence of the target gene of the Ricinus communis Cdc5-DBD protein in step (1) is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.
6.
3. The preparation method according to claim 1, characterized in that: The induced expression in step (2) is induced by culturing with 0.8 mM IPTG at 18°C and 160 rpm for 12 h.
4. The preparation method according to claim 1, characterized in that: In step (3), the protease inhibitor is PMSF; the reducing agent is DTT.
5. The preparation method according to claim 1, characterized in that: The affinity chromatography buffer in step (3) has a formula of 20 mM MES, 150 mM NaCl, 1 mM DTT, pH 6.
0.
6. The preparation method according to claim 1, characterized in that: The formula of the molecular sieve chromatography buffer in step (4) is 20mM MES, 150mM NaCl, pH 6.
0.
7. A method for crystallizing castor Cdc5-DBD protein, characterized in that: The castor Cdc5-DBD protein prepared by the method according to any one of claims 1 to 6 is prepared into a protein solution with a concentration of 5 mg / mL using a buffer solution, and then statically cultured in a 16° C. constant temperature incubator for 7 days in the presence of an MPD precipitant to obtain a single crystal of the castor Cdc5-DBD protein.
8. The crystallization method according to claim 7, characterized in that The MPD precipitant is an MPD aqueous solution, wherein the volume concentration of the MPD aqueous solution is 10-30%.
9. The crystallization method according to claim 7, characterized in that The formula of the buffer is 20 mM MES, 150 mM NaCl, pH 6.0.