A method for preparing crystals of cysteine ​​desulfurase

Abstract

The present invention belongs to the technical field of protein crystal preparation, and specifically relates to a method for preparing a crystal of cysteine ​​desulfurase. The present invention provides a method for preparing a crystal of cysteine ​​desulfurase, comprising hydrolyzing a cysteine ​​desulfurase protein by protease to obtain a cysteine ​​desulfurase domain; purifying the cysteine ​​desulfurase domain to obtain a purified cysteine ​​desulfurase domain; and concentrating the cysteine ​​desulfurase domain and then culturing the crystal to obtain its protein crystal. The present invention hydrolyzes the cysteine ​​desulfurase protein with a protease, which can hydrolyze the disordered region in the full-length protein, which is more conducive to the crystallization of the cysteine ​​desulfurase, and the activity of the cysteine ​​desulfurase protein obtained by the enzymatic hydrolysis is the same as that of the cysteine ​​desulfurase protein that has not been hydrolyzed, providing structural information and basis for the functional study of the cysteine ​​desulfurase protein.

Description

Technical Field

[0001] The invention belongs to the technical field of protein crystal preparation, and particularly relates to a method for preparing cysteine ​​desulfurase crystals. Background Art

[0002] Proteins are biological macromolecules composed of one or more peptide chains. Proteins are involved in every cell and all important components of the body. Cysteine ​​desulfurase is a class of enzymes that rely on pyridoxal phosphate. It is a homodimer that can catalyze the desulfurization of L-cysteine ​​to produce L-alanine, and at the same time transfer the activated sulfur atom to the cysteine ​​residue in its own active site to form Cys-SS, playing an important role in the formation of iron-sulfur clusters. When preparing crystals of cysteine ​​desulfurase, the full-length cysteine ​​desulfurase protein includes a disordered region at the N-terminus, which leads to significant protein degradation, becoming a major problem in preparing cysteine ​​desulfurase crystals. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a method for preparing crystals of cysteine ​​desulfurase. The method provided by the present invention uses trypsin to hydrolyze cysteine ​​desulfurase protein, which can enzymatically digest the disordered region, and the undigested stable part can be effectively used for the crystallization of cysteine ​​desulfurase, and the cysteine ​​desulfurase protein obtained by enzymatic hydrolysis has the same activity as the unhydrolyzed protein.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing crystals of cysteine ​​desulfurase, comprising the following steps:

[0006] The cysteine ​​desulfurase protein is subjected to protease hydrolysis to obtain the cysteine ​​desulfurase domain;

[0007] The cysteine ​​desulfurase domain is subjected to crystallization treatment to obtain crystals of the cysteine ​​desulfurase.

[0008] Preferably, the protease comprises trypsin.

[0009] Preferably, the amount of trypsin used is 1 μL of 0.05% trypsin-EDTA digestion solution per mg of cysteine ​​desulfurase protein.

[0010] Preferably, the protease hydrolysis temperature is 4°C and the time is 2 hours.

[0011] Preferably, the crystallization treatment includes purification treatment, concentration treatment and crystal culture.

[0012] Preferably, the crystal culture method includes vapor diffusion method, hanging drop method or sitting drop method.

[0013] Preferably, the method for preparing the cysteine ​​desulfurase protein comprises the following steps:

[0014] Transforming a recombinant expression vector containing a gene encoding cysteine ​​desulfurase into competent Escherichia coli cells, and screening to obtain a positive strain containing the recombinant expression vector;

[0015] The plasmid of the positive strain is extracted, and the extracted plasmid is transferred into mycobacterium competent cells, and induction culture, induced expression and purification are performed in sequence to obtain the cysteine ​​desulfurase protein.

[0016] Preferably, the initial vector in the recombinant expression vector includes a pMV-261 vector; the pMV-261 vector includes a flag tag.

[0017] Preferably, the induced expression comprises: cooling the bacterial solution obtained after the induction culture to 12°C to 16°C, adding acetamide and pyridoxal phosphate, and culturing for 3 to 5 days; the mass volume percentage of the acetamide is 0.1% to 0.2%; the concentration of the pyridoxal phosphate is 0.1 to 0.2 mM.

[0018] Beneficial effects:

[0019] The present invention provides a method for preparing a crystal of cysteine ​​desulfurase, comprising hydrolyzing a cysteine ​​desulfurase protein by protease to obtain a cysteine ​​desulfurase domain; purifying the cysteine ​​desulfurase domain to obtain a purified cysteine ​​desulfurase domain; and concentrating the cysteine ​​desulfurase domain and then culturing the crystal to obtain a protein crystal thereof. The present invention hydrolyzes the cysteine ​​desulfurase protein by protease, which can hydrolyze the disordered region in the full-length protein, and is more conducive to the crystallization of the cysteine ​​desulfurase. The activity of the cysteine ​​desulfurase protein obtained by the enzymatic hydrolysis is the same as that of the cysteine ​​desulfurase protein that has not been hydrolyzed, and provides structural information and a basis for the functional study of the cysteine ​​desulfurase protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 Schematic diagram of the sitting drop crystallization of cysteine ​​desulfurase in Example 1;

[0022] Figure 2 The results of gel filtration chromatography analysis (A) and desulfurization activity analysis (B) of Mycobacterium smegmatis cysteine ​​desulfurase (MSMEG_4538) before and after enzyme cleavage in Example 1 are shown. DETAILED DESCRIPTION

[0023] The present invention provides a method for preparing crystals of cysteine ​​desulfurase, comprising the following steps:

[0024] The cysteine ​​desulfurase protein is subjected to protease hydrolysis to obtain a hydrolyzed protein;

[0025] The hydrolyzed protein is subjected to crystallization treatment to obtain crystals of the cysteine ​​desulfurase.

[0026] The present invention preferably prepares cysteine ​​desulfurase protein by in vitro expression, and the preparation method of cysteine ​​desulfurase protein comprises:

[0027] The genomic DNA of wild-type Mycobacterium smegmatis was used as a template for PCR amplification to obtain the DNA fragment of cysteine ​​desulfurase;

[0028] Connecting the DNA fragment of cysteine ​​desulfurase with the modified shuttle pMV-261 fusion expression vector to obtain a recombinant expression vector;

[0029] The recombinant expression vector is transferred into a competent state to induce expression, thereby obtaining the cysteine ​​desulfurase protein.

[0030] The present invention preferably uses the genomic DNA of wild-type Mycobacterium smegmatis as a template for PCR amplification to obtain a PCR amplification product. The PCR amplification primers of the present invention are preferably designed using the gene encoding cysteine ​​desulfurase (MSMEG_4538) as a template; the upstream primer of the PCR amplification primer is 261-SufS-F, the nucleotide sequence of which is shown in SEQ ID NO.1, specifically: 5'-GAGAAAGGGAGTCCACATGatgagtaccagtgagtaccg-3'; the downstream primer of the PCR amplification primer is 261-SufS-R, the nucleotide sequence of which is shown in SEQ ID NO.2, specifically: 5'-GTTAACTACGTCGACTTActaCTTATCGTCGTCATCCTTGTAATCgaccgagccctcggcgat-3'.

[0031] The amplification system of the PCR of the present invention is preferably as shown in Table 1, and the amplification program is preferably as shown in Table 2.

[0032] Table 1 PCR amplification system

[0033] PCR components Volume / μL <![CDATA[ddH2O]]> 17 2×Phanta Max buffer 25 Upstream primer 261-SufS-F (10 μM) 1 Downstream primer 261-SufS-R (10 μM) 1 <![CDATA[Mycobacterium smegmatis mc 2 155) genomic DNA]]> 2 dNTP Mix (10mM) 2 DMSO 1 Phanta Max Super-Fidelity DNA Polymerase(Vazyme P505-d1) 1

[0034] Table 2 PCR amplification program

[0035]

[0036]

[0037] After obtaining the DNA fragment encoding cysteine ​​desulfurase, the present invention connects the DNA fragment of cysteine ​​desulfurase with the pMV-261 fusion expression vector to obtain a recombinant expression vector. When the present invention constructs the recombinant expression vector, the nucleotide sequence of the upstream primer 261-F used for linearizing the pMV-261 vector is preferably as shown in SEQ ID NO.3, specifically: 5'-TAAGTCGACGTA GTTAACTAGCGTACGATCGACTGC-3', and the nucleotide sequence of the downstream primer 261-R is preferably as shown in SEQ ID NO.4, specifically: 5'-CATGTGGACTCCCTTTCTCTTATCGGGTGGT GGC-3'. In the process of constructing the recombinant expression vector, the flag tag is connected to the carbon terminus of the cysteine ​​desulfurase by the above-mentioned PCR amplification and linearization connection. After obtaining the recombinant expression vector, the present invention preferably transfers the recombinant expression vector into Escherichia coli competent cells.

[0038] After the recombinant expression vector is transferred into the competent cells of Escherichia coli, the present invention preferably screens positive monoclonal strains to obtain positive strains containing the recombinant expression vector. The screening method of the present invention preferably comprises: applying the competent cells of Escherichia coli to a solid LB medium containing kanamycin for culturing to obtain a positive strain containing the recombinant expression vector. The concentration of kanamycin of the present invention is preferably 50 μg / mL. The present invention has no particular limitation on the method of culturing, and the culture can be carried out according to the conventional method of positive monoclonal screening in the art.

[0039] After obtaining the positive strain containing the recombinant expression vector, the present invention preferably extracts the plasmid of the positive strain containing the recombinant expression vector and performs sequencing to obtain a recombinant plasmid that is successfully sequenced. The present invention has no particular limitation on the extraction and sequencing methods, and conventional extraction and sequencing methods in the art can be used.

[0040] After obtaining the recombinant plasmid successfully sequenced, the present invention preferably transfers the recombinant plasmid successfully sequenced into mycobacterium competent cells mc 2 155, and induction culture is performed. The present invention preferably uses an electroporator to perform the transfer, and the parameter settings of the electroporator are preferably: the electroporation program selects Time constant protocol, the voltage is 2500V, the time is 5.7ms, and the thickness of the electroporation cup is 2mm. The brand of the GenePulser Xcell electroporator of the present invention is BIO-RAD. The mycobacterium of the present invention is preferably Mycobacterium smegmatis.

[0041] The induction culture of the present invention preferably includes monoclonal culture and expansion culture. The monoclonal culture of the present invention preferably includes applying the mycobacterium competent cells to a solid LB medium containing kanamycin and carbenicillin for monoclonal culture after 2 hours of recovery to obtain monoclonal bacterial cells. The temperature of the monoclonal culture of the present invention is preferably 37°C; the time is preferably 2 to 3 days. The concentrations of kanamycin and carbenicillin of the present invention are preferably 50 μg / mL.

[0042] The expanded culture of the present invention preferably includes picking the monoclonal bacteria and inoculating them into 5 mL of liquid LB medium containing kanamycin, carbenicillin and Tween 80 for the first expanded culture, and then transferring them to 1 L of liquid LB medium containing kanamycin, carbenicillin and Tween 80 for the second expanded culture, and culturing until the bacterial solution concentration reaches OD 600 =0.6 to 0.8, and the bacterial solution concentration is more preferably OD 600 =0.8. The temperature of the first enlarged culture of the present invention is preferably 37°C, and the time is preferably 24-36h; the temperature of the second enlarged culture is preferably 37°C. The concentrations of kanamycin and carbenicillin of the present invention are preferably 50 μg / mL, and the volume percentage of Tween 80 is preferably 0.01%.

[0043] After completing the induction culture, the present invention preferably induces expression in the bacterial solution obtained after the induction culture, and collects the mycobacterial cells. The induced expression of the present invention preferably includes cooling the bacterial solution obtained by the induction culture to 16°C, adding acetamide and pyridoxal phosphate for induced expression culture, inducing the expression of the gene encoding cysteine ​​desulfurase, and collecting the mycobacterial cells. The temperature of the induced expression culture of the present invention is preferably 12°C to 16°C, more preferably 16°C, and the time is preferably 3 to 5d, more preferably 3d. The mass volume percentage of acetamide in the present invention is preferably 0.1% to 0.2%, more preferably 0.2%, and the acetamide can induce the expression of DNA encoding cysteine ​​desulfurase; the concentration of the pyridoxal phosphate is preferably 0.2mM, and the pyridoxal phosphate serves as an auxiliary factor for induced expression.

[0044] After the induced expression is completed, the present invention preferably uses the collected mycobacterium cells for purification of cysteine ​​desulfurase protein to obtain the cysteine ​​desulfurase protein. The amino acid sequence of the cysteine ​​desulfurase protein of the present invention is shown in SEQ ID NO.6, which is as follows: The amino acid sequence of the full-length cysteine ​​desulfurase: MSTSEYRSLNAESDLPISADELSALATQLFAAGIRPGPDTPPQTAP VAPRGNVPGTAHPVAPPTEFVAVPSAVWSVPAHTSPVPEPVAPQTDPLVPPGVADLTAFTVPGGIVPTLPGVLAGARTAVPAAESPDYYFLRDTRAHQPVPQFPDRHEIFDVNAIRADFPILRETVNGKP LIWFDNAATTQKPQAVIDRLSHFYAHENSNIHRAAHELAARATDAYEDAREAVRRFIGAAKAEEIIFLRGTTEAINLVAHAWGGKHLGPGDEIVITHLEHHANIVPWQLLSRQTGAVLKVAPVDDAGNLLL GEFEDLLGPRTKLVAATQVSNALGTVTPVETIVELGHRYGARVLIDGAQSIPHMPIDVAALGADFFVFSGHKIYGPTGIGVLYGREDALAETPPWQGGGNMIADVTLERSLYQGPPNKFEAGTGNIADAV GLGEAIRYVERVGIERIAAYEHALLEYATPRLAAIPGVRIVGTAQEKASVLSFVLAGHEPLEVGKALNAEGIAVRAGHHCAQPILRRMGLEATVRPSFAFYNTFDEIDVFIDAVRRIAEGSVDYKDDDDK.

[0045] The purification of the present invention preferably includes: resuspending the collected mycobacterium cells with a binding buffer, performing high-pressure crushing and high-speed centrifugation to separate and obtain a supernatant; adding the obtained supernatant to an affinity chromatography column containing a flag antibody medium for chromatography; after the column flow-through is completed, washing 5 column volumes with a binding buffer, and then eluting the target protein with an elution buffer to obtain an elution sample; concentrating the obtained elution sample with an ultrafiltration tube to obtain a concentrated solution; and further purifying the obtained concentrated solution to obtain the cysteine ​​desulfurase protein.

[0046] The binding buffer of the present invention is preferably a mixture of 20mM Tris-HCl and 200mM NaCl, and the pH value is 7.4. The pressure of the high-pressure crushing of the present invention is preferably 800-1200 bar, and more preferably 1000 bar. The speed of the high-speed centrifugation is preferably 18000 rpm (26500×g), and the centrifugation time is preferably 40 min. The elution buffer of the present invention is preferably a mixture of 20mM Tris-HCl, 200mM NaCl and 200μg / mL DYKDDDDK polypeptide, and the pH value is preferably 7.4. The molecular weight cutoff of the ultrafiltration tube of the present invention is preferably 100kDa.

[0047] The further purification of the present invention preferably uses a gel filtration chromatography column, and the pre-packed column of the gel filtration chromatography column is preferably Superdex 200Increase 10 / 300GL.

[0048] After obtaining the cysteine ​​desulfurase protein, the present invention performs protease hydrolysis on the cysteine ​​desulfurase protein to obtain the cysteine ​​desulfurase domain. The protease of the present invention is preferably trypsin; the amount of the protease is preferably 1 μL of 0.05% trypsin-EDTA digestion solution per mg of cysteine ​​desulfurase protein; the present invention does not specifically limit the source of the 0.05% trypsin-EDTA digestion solution, and a conventional commercial product in the art can be used, such as Gibco T M 25300054. The hydrolysis temperature of the present invention is preferably 4°C, and the hydrolysis time is preferably 2h.

[0049] After obtaining the cysteine ​​desulfurase domain, the present invention preferably purifies the cysteine ​​desulfurase domain; the purification treatment of the present invention preferably includes purifying the hydrolyzed protein by gel filtration chromatography. The present invention preferably uses gel filtration chromatography for the purification, and the gel filtration chromatography column used in the gel filtration chromatography is preferably Superdex 200Increase 10 / 300GL.

[0050] After the purification treatment, the present invention preferably uses an ultrafiltration tube to concentrate the purified cysteine ​​desulfurase domain, and the molecular weight cutoff of the ultrafiltration tube is preferably 50 kDa.

[0051] After the concentration, the present invention preferably performs a crystallization treatment on the concentrate obtained by the concentration to obtain crystals of cysteine ​​desulfurase. The method of the crystallization treatment of the present invention preferably includes a vapor diffusion method, a hanging drop method or a sitting drop method, and more preferably a sitting drop method. The present invention has no special limitation on the operation of the sitting drop method, and a conventional sitting drop method can be used.

[0052] The method for preparing cysteine ​​desulfurase protein crystals provided by the present invention uses protease to enzymatically hydrolyze the cysteine ​​desulfurase protein, which can enzymatically hydrolyze the disordered regions in the whole protein, which is more conducive to the crystallization of cysteine ​​desulfurase, and the activity of the cysteine ​​desulfurase protein obtained by enzymatic hydrolysis is the same as that of the cysteine ​​desulfurase protein that has not been enzymatically hydrolyzed, providing structural information and basis for the functional research of cysteine ​​desulfurase protein.

[0053] In order to further illustrate the present invention, the method for preparing the crystal of cysteine ​​desulfurase provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] A method for preparing crystals of cysteine ​​desulfurase, the specific steps are as follows:

[0056] 1) Obtaining DNA sequence encoding cysteine ​​desulfurase protein

[0057] The PCR amplification primers were designed using the DNA sequence encoding cysteine ​​desulfurase protein (SEQ ID NO.5) as a template, as follows:

[0058] Upstream primers: 261-SufS-F: GAGAAAGGGAGTCCACATGatgagtaccagtgagtaccg and 261-F: TAAGTCGACGTAGTTAACTAGCGTACGATCGACTGC;

[0059] Downstream primers: 261-SufS-R: GTTAACTACGTCGACTTActaCTTATCGTCGTCATCCTTGTAATCgaccgagccctcggcgat and 261-R: CATGTGGACTCCCTTTCTCTTA TCGGGTGGTGGC.

[0060] Wild-type Mycobacterium smegmatis 2155) genomic DNA (accession number in Mycobrowser (Mycobacterial browser) is MSMEG_4358) as a template for PCR amplification. The PCR amplification system and amplification program are shown in Tables 1 and 2, respectively.

[0061] Table 1 PCR amplification system

[0062] PCR components Volume / μL <![CDATA[ddH2O]]> 17 2xPhanta Max buffer 25 Upstream primer 261-SufS-F or 261-F (10 μM) 1 Downstream primer 261-SufS-R or 261-R (10 μM) 1 <![CDATA[Mycobacterium smegmatis mc 2 155) genomic DNA]]> 2 dNTP Mix (10mM) 2 DMSO 1 Phanta Max Super-Fidelity DNA Polymerase(Vazyme P505-d1) 1

[0063] Table 2 PCR amplification program

[0064]

[0065] Finally, a 741 bp DNA sequence encoding cysteine ​​desulfurase protein was obtained (SEQ ID NO.5);

[0066] 2) Construction of gene recombinant expression vector

[0067] The linearized pMV-261 vector was connected to the DNA sequence encoding the cysteine ​​desulfurase protein obtained in step 1), and transformed into Escherichia coli DH5α competent cells by heat stimulation; the Escherichia coli DH5α competent cells were spread on a solid LB medium containing 50 μg / mL kanamycin for screening to obtain a positive strain containing the recombinant expression vector;

[0068] The primers used for linearization of pMV-261 vector are:

[0069] Upstream primer: 261-F: TAAGTCGACGTAGTTAACTAGCGTACGATCGACTGC;

[0070] Downstream primer: 261-R: CATGTGGACTCCCTTTCTCTTATCGGGTGGTGGC.

[0071] The amplification system is the same as that in Table 1, and the amplification procedure is the same as that in Table 2.

[0072] 3) Extraction of plasmids from positive strains and sequencing

[0073] The extraction of pMV-261-SufS-C-flag recombinant plasmid in Escherichia coli is based on the principle of alkaline lysis method, and the mature Tiangen kit on the market is used for extraction. The specific steps are: the first step is to add 1-5mL of bacterial solution to a centrifuge tube, centrifuge at 12000rpm (13400xg) for 1min, and collect the precipitate. The second step is to add 250μL of solution P1 (containing RnaseA) to the precipitate to suspend the bacterial precipitate. The third step is to add 250μL of solution P2 to the centrifuge tube, gently turn it upside down 6-8 times to fully lyse the bacteria. The fourth step is to add 250μL of solution P3 to the centrifuge tube, immediately turn it upside down 6-8 times, mix it thoroughly, and centrifuge it at 12000rpm for 10min after the white precipitate appears. The fifth step is to transfer the supernatant collected in the previous step to the adsorption column, centrifuge it at 12000rpm for 1min, pour out the waste liquid and put the adsorption column into the collection tube. Step 6: Add 600 μL of rinse solution to the adsorption column and centrifuge at 12,000 rpm for 1 min. Repeat this step twice. Step 7: Place the adsorption column in a clean centrifuge tube, add 50 μL of 65°C preheated distilled water to the adsorption column, place at room temperature for 2 min, then centrifuge at 12,000 rpm for 2 min, and collect the plasmid solution into the centrifuge tube. Next, send the extracted plasmid to Genewise Sequencing Company for sequencing to obtain a recombinant plasmid consistent with the expected sequence for subsequent recombinant expression.

[0074] 4) Inducible expression of cysteine ​​desulfurase protein

[0075] The recombinant plasmid successfully sequenced in step 3) was transformed into Mycobacterium smegmatis by electroporation. 2 After 2 h of recovery, the competent cells were spread on solid LB medium containing 50 μg / mL kanamycin and 50 μg / mL carbenicillin, and monoclonal culture was performed at 37°C for 2 to 3 days to obtain monoclonal bacteria; the monoclonal bacteria were picked and inoculated into liquid LB medium (5 mL) containing 50 μg / mL kanamycin, 50 μg / mL carbenicillin and 0.01% Tween 80 and cultured for 24 to 48 h, and then transferred to liquid LB medium (1 L) containing 50 μg / mL kanamycin, 50 μg / mL carbenicillin and 0.01% Tween 80 for expansion culture until the concentration of the bacterial solution was OD 600 =0.8; then the cultured bacterial solution was cooled to 16°C, 0.2% (w / v) acetamide was added to the culture medium to induce the expression of the DNA encoding cysteine ​​desulfurase, and 0.2 mM pyridoxal phosphate was added as a cofactor, and the cells were collected after culturing at 16°C for 3 to 5 days;

[0076] 5) Purification of cysteine ​​desulfurase protein

[0077] The bacteria collected in step 4) were resuspended with binding buffer (20mM Tris-HCl pH 7.4, 200mM NaCl), and high-pressure crushing was performed at a pressure of 1000 bar; the crushed bacteria were centrifuged at a speed of 18000 rpm for 40 min, and the obtained supernatant was added to an affinity chromatography column containing an anti-flag antibody medium. After the column flow-through was completed, five column volumes were rinsed with binding buffer, and then the target protein was eluted with elution buffer (20mM Tris-HCl pH 7.4, 200mM NaCl, 200μg / mL DYKDDDDK); the eluted sample was concentrated using an ultrafiltration tube with a molecular weight cutoff of 100kDa; the concentrated eluted sample was further purified using a gel filtration column (Superdex 200Increase10 / 300GL column) to obtain a cysteine ​​desulfurase protein (the protein sequence is shown in SEQ ID NO.6);

[0078] 6) Crystallization of Cysteine ​​Desulfurase Protein

[0079] The protein purified in step 5) was added with 1 μL of trypsin-EDTA (0.05%) (Gibco TM 25300054). Hydrolyze at 4℃ for 2h; put the hydrolyzed protein into the gel filtration column (Superdexe200Increase 10 / 300GL column) for re-purification; concentrate the re-purified protein using an ultrafiltration tube with a molecular weight cutoff of 50kDa, and use analytical ultracentrifugation to analyze the aggregation state of the protein; use the sitting drop method to culture the concentrated protein for crystallization, the specific steps are: add 80μL of pool solution, that is, protein solvents of different formulas, to the reservoir, add 500nL of protein solution (6mg / mg and 12mg / mg) that has been enzymatically hydrolyzed, purified and concentrated to the Drop of the sitting drop plate, and spot 500nL of pool solution (0.1mM Bis-Tris pH6.5, 9-24% w / v PEG10000) on the protein, seal it with a sealing film with high transparency, place the sitting drop plate in an incubator for culture, set the culture temperature to 16℃, and the time is 7d. Sitting drop method as Figure 1 shown.

[0080] The activity of the protein hydrolyzed in step 6) of Example 1 was determined by the methylene blue colorimetric method. The specific steps are: first, Na2S was used to make a comparison between the methylene blue light absorption value and S 2-The standard curve of concentration. Next, the desulfurase activity of SufS was determined: first, 3 μL of cysteine ​​desulfurase was added to a 1.5 mL centrifuge tube, and then different concentrations of L-Cys substrate were added to the reaction system (the final concentrations were 150 μM, 75 μM, 37.5 μM, 18.75 μM, 9.38 μM, 4.69 μM, 2.34 μM, 1.17 μM, 0.59 μM, 0 μM), and the reaction was carried out at 25 ° C for 3 minutes; after the reaction, N, N-dimethyl-p-phenylenediamine (DMPD) (prepared with 7.2 M hydrochloric acid solution) and FeCl3 (prepared with 1.2 M hydrochloric acid solution) were added to the reaction system to terminate the reaction, and incubated in the dark for 30 minutes to generate methylene blue; then centrifuged at 12000 rpm for 10 minutes, and the supernatant was taken out and added to a transparent 96-well plate for light absorption measurement. The S released by the reaction was determined by measuring the light absorption of methylene blue at 670 nm. 2- The reaction was repeated at least three times. The experimental data were processed using GraphpadPad software to fit the Michaelis-Menten curve. The error bar at each point represents the standard deviation. Figure 2 .

[0081] Depend on Figure 2 The results showed that the activity of the cysteine ​​desulfurase protein after trypsin hydrolysis was the same as that of the full-length cysteine ​​desulfurase protein.

[0082] It can be seen from the above embodiments that the method provided by the present invention uses protease to enzymatically hydrolyze cysteine ​​desulfurase protein, which can enzymatically hydrolyze the disordered region in the full-length protein, which is more conducive to the crystallization of cysteine ​​desulfurase. It can achieve the situation that no crystal growth occurs without excision, and the activity of the cysteine ​​desulfurase protein obtained by enzymatic hydrolysis is the same as that of the cysteine ​​desulfurase protein that has not been enzymatically hydrolyzed, providing structural information and basis for the functional study of cysteine ​​desulfurase protein.

[0083] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing crystals of cysteine ​​desulfurase, characterized in that: The following steps are involved: The cysteine ​​desulfurase protein is subjected to protease hydrolysis to obtain a cysteine ​​desulfurase domain, wherein the amino acid sequence of the cysteine ​​desulfurase protein is shown in SEQ ID NO.6; performing a crystallization treatment on the cysteine ​​desulfurase domain to obtain a crystal of the cysteine ​​desulfurase; The protease is trypsin; the amount of trypsin used is 1 μL of 0.05% trypsin-EDTA digestion solution per mg of cysteine ​​desulfurase protein; The crystallization process includes purification, concentration and crystal culture; After the purification treatment, the purified cysteine ​​desulfurase domain is concentrated using an ultrafiltration tube, and the molecular weight cutoff of the ultrafiltration tube is 50 kDa.

2. The preparation method according to claim 1, characterized in that: The temperature of the protease hydrolysis is 4°C and the time is 2h.

3. The preparation method according to claim 1, characterized in that: The crystal culture method includes a vapor diffusion method, a hanging drop method or a sitting drop method.

4. The preparation method according to claim 1, characterized in that: The preparation method of the cysteine ​​desulfurase protein comprises the following steps: Transforming a recombinant expression vector containing a gene encoding cysteine ​​desulfurase into competent Escherichia coli cells, and screening to obtain a positive strain containing the recombinant expression vector; The plasmid of the positive strain is extracted, and the extracted plasmid is transferred into mycobacterium competent cells, and induction culture, induced expression and purification are performed in sequence to obtain the cysteine ​​desulfurase protein.

5. The preparation method according to claim 4, characterized in that: The initial vector in the recombinant expression vector includes a pMV-261 vector; the pMV-261 vector includes a flag tag.

6. The preparation method according to claim 4, characterized in that: The induction expression comprises: cooling the bacterial solution obtained after the induction culture to 12-16°C, adding acetamide and pyridoxal phosphate, and culturing for 3-5 days; the mass volume percentage of the acetamide is 0.1%-0.2%; the concentration of the pyridoxal phosphate is 0.1-0.2mM.

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