A method for expressing a specific domain of Mouse SPO11 protein

Abstract

The invention discloses a method for expressing a specific domain of a Mouse SPO11 protein. The method comprises the following steps: using a sequence of the Mouse SPO11 protein as a template, designing primers for PCR amplification, and obtaining a Mouse SPO11 gene fragment after PCR amplification; in an Alp_PET_StHis_SUMO empty vector, sequentially designing a Strep tag II affinity tag, an 8x his affinity tag, and a SUMO solubilizing tag at the N-terminus of a multiple cloning site; subjecting the Mouse SPO11 gene fragment after PCR amplification to gel electrophoresis, followed by gel excision recovery, and then simultaneously subjecting the Mouse SPO11 gene fragment to BamHI and EcoRI double enzyme digestion with the expression vector Alp_PET_StHis_SUMO, and then subjecting the Mouse SPO11 gene fragment to gel excision recovery, connection, transformation, colony PCR, colony culture, and plasmid extraction to obtain a successfully constructed plasmid; inducing expression of the plasmid to obtain an expressed bacterial solution; and purifying the expressed bacterial solution to obtain the Mouse SPO11 protein specific domain. The invention can express the specific structural domain of Mouse SPO11 protein and obtain high-purity Mouse SPO11 protein.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a method for expressing a specific domain of Mouse SPO11 protein. Background Art

[0002] The E. coli expression system has the advantages of low cost and rapid expression. However, the E. coli expression system lacks appropriate post-translational processing mechanisms; therefore, the target protein is prone to three-dimensional folding errors to form inclusion bodies, which makes subsequent purification and recovery difficult. Now, the study of the physiological function of the SPO11 gene is a hot topic at the forefront. Because this helps to gain a deeper understanding of the mechanism of germ cell development and provide an important reference for the diagnosis and treatment of related diseases. Despite this, the structural and functional research related to SPO11 is limited by the acquisition of SPO11 protein. So far, there is no Mouse SPO11 protein for sale on the market, indicating that there are technical difficulties in the expression and purification of this protein.

[0003] In summary, after extensive searching by the applicant, it is found in the art that at least Mouse SPO11 protein is difficult to express and purify. Therefore, it is necessary to develop or improve a method for expressing a specific domain of Mouse SPO11 protein. Summary of the invention

[0004] Based on this, in order to solve the problem that Mouse SPO11 protein is difficult to express and purify, the present invention provides a method for expressing a specific domain of Mouse SPO11 protein, and the specific technical scheme is as follows:

[0005] A method for expressing a specific domain of Mouse SPO11 protein, comprising the following steps:

[0006] Using the Mouse SPO11 protein sequence as a template, primers are designed for PCR amplification to obtain the Mouse SPO11 gene fragment after PCR amplification;

[0007] In the Alp_PET_StHis_SUMO empty vector, Strep tag II affinity tag, 8x his affinity tag and SUMO solubility tag were designed in sequence at the N-terminus of the multiple cloning site to obtain the expression vector Alp_PET_StHis_SUMO;

[0008] The Mouse SPO11 gene fragment amplified by PCR is subjected to gel electrophoresis, and then the gel is cut and recovered, and then the fragment is double-digested with BamHI and EcoRI at the same time as the expression vector Alp_PET_StHis_SUMO to obtain a double-digested fragment and a double-digested vector;

[0009] The double-enzyme-digested fragment and the double-enzyme-digested vector are excised and recovered by gel excision, and then ligated using T4 ligase to obtain a ligation product, wherein the molar amount of the double-enzyme-digested fragment and the molar amount of the double-enzyme-digested vector is 4 to 6:1;

[0010] All the ligation products were transformed into DH5α competent cells, and after transformation, all the transformation products were spread on a Kana resistance LB solid plate with a final concentration of 30-70 μg / mL to obtain a ligation single colony;

[0011] The connected single colony is subjected to colony PCR, and then inoculated into a Kana resistance LB liquid culture medium with a concentration of 30 to 70 μg / mL for colony culture to obtain a PCR bacterial solution, and the PCR bacterial solution is subjected to plasmid extraction and then sequencing. If the result is correct, a successfully constructed plasmid is obtained;

[0012] Inducing the expression of the plasmid to obtain an expressed bacterial solution;

[0013] The expressed bacterial solution is purified to obtain the specific structural domain of the Mouse SPO11 protein.

[0014] Furthermore, the inducing expression comprises the following steps:

[0015] The plasmid is transformed into the competent E. coli expression strain CODON PLUS, and then the transformation product is spread on a KANA resistance LB solid plate with a final concentration of 30-70 μg / mL, and then cultured at 35-39° C. for 16-24 hours to obtain a single plasmid colony;

[0016] Take a single colony of the plasmid and inoculate it in a KANA resistance LB liquid medium with a final concentration of 30-70 μg / mL, then culture it at 35-39°C for 16-24h, and then expand the culture at 35-39°C until the OD 600 =0.6-0.8, then placed in tap water to cool to room temperature, then added with isopropyl-β-D-thiogalactoside at a final concentration of 0.5-1.0 mM, induced expression at 12-16° C. for 16-24 h, to obtain the expressed bacterial solution.

[0017] Further, the purification comprises the following steps:

[0018] The expressed bacterial solution is centrifuged at 3500-4500 rpm at 3-5°C, the precipitated bacterial bodies are collected, and then the precipitated bacterial bodies are crushed and lysed, and the supernatant is obtained by centrifugation after lysis, and the supernatant is subjected to Ni affinity chromatography, and then Strep affinity chromatography, and then enzymatic digestion with ULP1 enzyme, and then Ni affinity chromatography, to obtain the specific domain of the Mouse SPO11 protein.

[0019] Furthermore, the amino acid sequence of the Mouse SPO11 protein is:

[0020] AGATRVASSSEVLTAIENIIQDIIKSLARNEVPAFTIDNRSSWENIMFDDSVGLRMIPQC TTRKIRSDSPKSVKKFALILKVLSMIYKLIQSDTYATKRDIYYTDSQLFGNQAAVDSAIDDISC MLKVPRRSLHVLST.

[0021] Further, the primers for PCR amplification include a forward primer and a reverse primer; the forward primer is: 5'-GTCACCACGCGGATCCGCTGGTGCAACCCGTGTGGCAAG-3', and the reverse primer is: 5'-CCATCGACCGGAATTCTTAGGTACTCAGCACATGCAGGCTACGAC-3';

[0022] The conditions for the colony PCR are as follows: forward primer: 5'-ATGCCGGCCACGATGCGTC-3', reverse primer: 5'-CCGGATATAGTTCCTCCTTTC-3'.

[0023] Further, the double-digested fragment is

[0024] GGATCCGCTGGTGCAACCCGTGTGGCAAGCAGCAGTGAAGTGCTGACCGCAATTGAAAATATTATCCAGGATATCATCAAGAGCCTGGCACGCAATGAAGTTCCGGCATTCACTATTGATAACCGCAGCAGCTGGGAAAATATTATGTTTGATGACAGCGTGGGCCTGCGTATGATTCCGCAGTGTACCACCCGTAAAATTCGCAGCGATAGTCC GAAAAGCGTGAAAAAATTTGCACTGATTCTGAAGGTGCTGAGTATGATCTATAAGCTGATTCAGAGTGACACCTATGCAACCAAACGTGATATCTATTACACCGATAGCCAGCTGTTTGGCAATCAGGCAGCAGTTGATAGTGCAATTGATGATATTAGCTGCATGCTGAAAGTGCCGCGTCGTAGCCTGCATGTGCTGAGTACCTAAGAATTC.

[0025] Furthermore, the conditions of the gel electrophoresis are: 0.5-1.5% agarose, 25-35 min;

[0026] The double enzyme digestion conditions are 35-39°C, 16-24h;

[0027] The connection conditions are 15-17° C., 4-6 h;

[0028] The conditions for the colony culture are 35-39° C., 16-24 h, and 210-230 rpm.

[0029] Further, the Ni affinity chromatography includes a His affinity tag;

[0030] The Strep affinity chromatography includes a Strep II affinity tag.

[0031] Furthermore, the concentration of NaCl in the purification buffer system of the Ni affinity chromatography and the Strep affinity chromatography is 1000-1500 mM.

[0032] Furthermore, the lysis uses a lysis solution, which includes 1000-1500 mM NaCl, 20-50 mM Tris-HCL, 20-60 mM imidazole, 0.5-1.0 mM PMSF, 50-100 μg / ml DNaseI, 5-10% glycerol, 0.1-1.0% TritonX-100 and 1-2 mM MgCl2.

[0033] The present invention adds a SUMO solubility-promoting tag when expressing Mouse SPO11, thereby solving the problem of the target protein forming inclusion bodies in the large intestine expression system, and the SUMO tag increases the expression amount of the soluble target protein in the Escherichia coli expression system, helps the protein to fold correctly, and improves the protein stability; the present invention uses a high salt system, 1000-1500mM NaCl, in the purification process, to improve the stability and solubility of the Mouse SPO11 protein, and to improve the yield and purity of Mouse SPO11; the present invention designs a Strep II and his dual affinity tag, and removes non-target proteins as much as possible through two affinity chromatography, thereby being more conducive to obtaining a high-purity Mouse SPO11 protein; the ULP1 enzyme of the present invention can specifically recognize the SUMO tag, and the ULP1 enzyme has extremely high enzymatic cleavage efficiency, and can complete 95% fusion protein cleavage in a short time, thereby being more conducive to obtaining a high-purity target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the structure of Mouse SPO11 protein predicted by alphafold2. The circle is the target domain of SPO11 expressed and purified by the present invention, which is located outside the whole protein.

[0035] Figure 2 It is the SPO11 protein domain designed by the present invention predicted by alphafold2;

[0036] Figure 3 It is the result of 1% agarose gel electrophoresis of the product after double enzyme digestion;

[0037] Figure 4 It is the result of 1% agarose gel electrophoresis of the product after double enzyme digestion;

[0038] Figure 5 It is the double-enzyme digestion vector map;

[0039] Figure 6 It is the result of colony PCR electrophoresis;

[0040] Figure 7 It is the electrophoresis result of the sample during the purification process;

[0041] Figure 8 It is the electrophoresis result of the sample during the purification process;

[0042] Fig. 9 It is the result of induced expression of SPO11 fusion protein;

[0043] Fig.10 It is the result of induced expression of SPO11 fusion protein;

[0044] Fig.11 This is the SDS PAGE gel image of the purification process. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the protection scope of the present invention.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0047] A method for expressing a specific domain of Mouse SPO11 protein in one embodiment of the present invention comprises the following steps:

[0048] Using the Mouse SPO11 protein sequence as a template, primers are designed for PCR amplification to obtain the Mouse SPO11 gene fragment after PCR amplification;

[0049] In the Alp_PET_StHis_SUMO empty vector, Strep tag II affinity tag, 8x his affinity tag and SUMO solubility tag were designed in sequence at the N-terminus of the multiple cloning site to obtain the expression vector Alp_PET_StHis_SUMO;

[0050] The Mouse SPO11 gene fragment amplified by PCR is subjected to gel electrophoresis, and then the gel is cut and recovered, and then the fragment is double-digested with BamHI and EcoRI at the same time as the expression vector Alp_PET_StHis_SUMO to obtain a double-digested fragment and a double-digested vector;

[0051] The double-enzyme-digested fragment and the double-enzyme-digested vector are excised and recovered by gel excision, and then ligated using T4 ligase to obtain a ligation product, wherein the molar amount of the double-enzyme-digested fragment and the molar amount of the double-enzyme-digested vector is 4 to 6:1;

[0052] All the ligation products were transformed into DH5α competent cells, and after transformation, all the transformation products were spread on a Kana resistance LB solid plate with a final concentration of 30-70 μg / mL to obtain a ligation single colony;

[0053] The connected single colony is subjected to colony PCR, and then inoculated into a Kana resistance LB liquid culture medium with a concentration of 30 to 70 μg / mL for colony culture to obtain a PCR bacterial solution, and the PCR bacterial solution is subjected to plasmid extraction and then sequencing. If the result is correct, a successfully constructed plasmid is obtained;

[0054] Inducing the expression of the plasmid to obtain an expressed bacterial solution;

[0055] The expressed bacterial solution is purified to obtain the specific structural domain of the Mouse SPO11 protein.

[0056] In one embodiment, the inducing expression comprises the following steps:

[0057] The plasmid is transformed into the competent E. coli expression strain CODON PLUS, and then the transformation product is spread on a KANA resistance LB solid plate with a final concentration of 30-70 μg / mL, and then cultured at 35-39° C. for 16-24 hours to obtain a single plasmid colony;

[0058] A single colony of the plasmid was inoculated into a KANA-resistant LB liquid culture medium with a final concentration of 30 to 70 μg / mL, and then cultured at 35 to 39° C. for 16 to 24 hours, and then expanded at 35 to 39° C. to OD600 = 0.6 to 0.8, and then placed in tap water to cool to room temperature, and then 0.5 to 1.0 mM isopropyl-β-D-thiogalactoside was added, and expression was induced at 12 to 16° C. for 16 to 24 hours to obtain the expressed bacterial solution.

[0059] In one embodiment, the purification comprises the following steps:

[0060] The expressed bacterial solution is centrifuged at 3500-4500 rpm at 3-5°C, the precipitated bacterial bodies are collected, and then the precipitated bacterial bodies are crushed and lysed, and the supernatant is obtained by centrifugation after lysis, and the supernatant is subjected to Ni affinity chromatography, and then Strep affinity chromatography, and then enzymatic digestion with ULP1 enzyme, and then Ni affinity chromatography, to obtain the specific domain of the Mouse SPO11 protein.

[0061] In one embodiment, the amino acid sequence of the Mouse SPO11 protein is:

[0062] AGATRVASSSEVLTAIENIIQDIIKSLARNEVPAFTIDNRSSWENIMFDDSVGLRMIPQC TTRKIRSDSPKSVKKFALILKVLSMIYKLIQSDTYATKRDIYYTDSQLFGNQAAVDSAIDDISC MLKVPRRSLHVLST.

[0063] In one embodiment, the primers for PCR amplification include a forward primer and a reverse primer; the forward primer is: 5'-GTCACCACGCGGATCCGCTGGTGCAACCCGTGTGGCAAG-3', and the reverse primer is: 5'

[0064] -CCATCGACCGGAATTCTTAGGTACTCAGCACATGCAGGCTACGAC-3';

[0065] The conditions for the colony PCR are as follows: forward primer: 5'-ATGCCGGCCACGATGCGTC-3', reverse primer: 5'-CCGGATATAGTTCCTCCTTTC-3'.

[0066] In one embodiment, the double-digested fragment is

[0067] GGATCCGCTGGTGCAACCCGTGTGGCAAGCAGCAGTGAAGTGCTGACCGCAATTGAAAATATTA

[0068] TCCAGGATATCATCAAGAGCCTGGCACGCAATGAAGTTCCGGCATTCACTATTGATAACCGCAG

[0069] CAGCTGGGAAAATATTATGTTTGATGACAGCGTGGGCCTGCGTATGATTCCGCAGTGTACCACC

[0070] CGTAAAATTCGCAGCGATAGTCCGAAAAGCGTGAAAAAATTTGCACTGATTCTGAAGGTGCTGA

[0071] GTATGATCTATAAGCTGATTCAGAGTGACACCTATGCAACCAAACGTGATATCTATTACACCGA

[0072] TAGCCAGCTGTTTGGCAATCAGGCAGCAGTTGATAGTGCAATTGATGATATTAGCTGCATGCTGAAAGTGCCGCGTCGTAGCCTGCATGTGCTGAGTACCTAAGAATTC.

[0073] In one embodiment, the conditions of gel electrophoresis are: 0.5-1.5% agarose, 25-35 min;

[0074] The double enzyme digestion conditions are 35-39°C, 16-24h;

[0075] The connection conditions are 15-17° C., 4-6 h;

[0076] The conditions for the colony culture are 35-39° C., 16-24 h, and 210-230 rpm.

[0077] In one embodiment, the Ni affinity chromatography includes a His affinity tag;

[0078] The Strep affinity chromatography includes a Strep II affinity tag.

[0079] In one embodiment, the concentration of NaCl in the purification buffer system of the Ni affinity chromatography and the Strep affinity chromatography is 1000-1500 mM.

[0080] In one embodiment, the lysis uses a lysis solution, which includes 1000-1500 mM NaCl, 20-50 mM Tris-HCL, 20-60 mM imidazole, 0.5-1.0 mM PMSF, 50-100 μg / ml DNaseI, 5-10% glycerol, 0.1-1.0% TritonX-100 and 1-2 mM MgCl2.

[0081] The embodiments of the present invention will be described in detail below with reference to specific examples.

[0082] Embodiment 1:

[0083] Using the sequence containing Mouse SPO11 protein as a template, primers were designed for PCR amplification to obtain the Mouse SPO11 gene fragment after PCR amplification. The forward primer for PCR amplification was: 5'-GTCACCACGCGGATCCGCTGGTGCAACCCGTGTGGCAAG-3', and the reverse primer was: 5'-CCATCGACCGGAATTCTTAGGTACTCAGCACATGCAGGCTACGAC-3'. The PCR amplification system and reaction procedure are shown in Table 1. The amino acid sequence of Mouse SPO11 protein expression is: AGATRVASSSEVLTAIENIIQDIIKSLARNEVPAFTIDNRSSWENIMFDDSVGLRMIPQCTTRK IRSDSPKSVKKFALILKVLSMIYKLIQSDTYATKRDIYYTDSQLFGNQAAVDSAIDDISCMLKV PRRSLHVLST;

[0084] In the Alp_PET_StHis_SUMO empty vector, Strep tag II affinity tag, 8x his affinity tag and SUMO solubility tag were designed in sequence at the N-terminus of the multiple cloning site to obtain the expression vector Alp_PET_StHis_SUMO;

[0085] The Mouse SPO11 gene fragment amplified by PCR was subjected to 1% agarose gel electrophoresis (30 min), and then the gel was cut and recovered, and then the expression vector Alp_PET_StHis_SUMO was double-digested with BamHI and EcoRI at the same time. The double-digestion conditions were 37°C for 20 h to obtain the double-digested fragment and the double-digested vector. The double-digested fragment (429 bp after double-digestion) was GGATCCGCTGGTGCAACCCGTGTGGCAAGCAGCAGTGAAGTGCTGACCGCAATTGAAAATATTATCCAGGATATCATCAAGAGCCTGGCACGCAATGAAGTTCCGGCATTCACTATTGATAACCGCAGCAGCTGGGAAAATATTATGTTTGAT GACAGCGTGGGCCTGCGTATGATTCCGCAGTGTACCACCCGTAAAATTCGCAGCGATAGTCCGAAAAGCGTGAAAAAATTTGCACTGATTCTGAAGGTGCTGAGTATGATCTATAAGCTGATTCAGAGTGACACCTATGCAA CCAAACGTGATATCTATTACACCGATAGCCAGCTGTTTGGCAATCAGGCAGCAGTTGATAGTGCAATTGATGATATTAGCTGCATGCTGAAAGTGCCGCGTCGTAGCCTGCATGTGCTGAGTACCTAAGAATTC, the vector after double enzyme digestion is as follows Figure 5 As shown;

[0086] The double-enzyme-digested fragment and the double-enzyme-digested vector were excised and recovered from gel, and then incubated with T4 ligase at 16° C. for 5 h to obtain a ligation product, wherein the molar amount of the double-enzyme-digested fragment and the molar amount of the double-enzyme-digested vector was 5:1;

[0087] All the ligation products were transformed into DH5α competent cells, and after transformation, all the transformation products were spread on a Kana resistance LB solid plate with a final concentration of 50 μg / mL to obtain a ligation single colony;

[0088] The connected single colony was subjected to colony PCR, and then inoculated into 5 mL of 50 μg / mL Kana resistance LB liquid medium for colony culture. The PCR bacterial solution was cultured at 37°C and 220 rpm for 20 h. The PCR bacterial solution was subjected to plasmid extraction. The extraction kit was (Sangon Biotechnology, catalog number B518191-0050). Then sequencing was performed. After the result was correct, the successfully constructed plasmid was obtained. Among them, the forward primer of colony PCR was 5'-ATGCCGGCCACGATGCGTC-3', and the reverse primer was 5'-CCGGATATAGTTCCTCCTTTC-3'. The colony PCR system and reaction procedure are shown in Table 2. The colony PCR electrophoresis results are shown in Figure 6 As shown, the sequencing primers are

[0089] Forward primer T7: 5'-3': TAATACGACTCACTATAGGG Reverse primer T7ter: 5'-3': TGCTAGTTATTGCTCAGCGG The sequencing results are as follows, the results are correct, and the plasmid is successfully constructed.

[0090]

[0091]

[0092] The correctly sequenced plasmid was transformed into the competent Escherichia coli expression strain CODON PLUS, and then the transformation product was spread on a KANA-resistant LB solid plate with a final concentration of 50 μg / mL, and then cultured at 37°C for 20 hours to obtain a single plasmid colony; a single plasmid colony was inoculated in 5 mL of a KANA-resistant LB liquid culture medium with a final concentration of 50 μg / mL, and then cultured at 37°C and 220 rpm for 20 hours, and then 1 mL of the liquid culture medium in the previous step was inoculated in 1 L of a KANA-resistant LB liquid culture medium with a final concentration of 50 μg / mL for expansion culture, cultured at 37°C and 220 rpm to OD600=0.7, and then placed in tap water to cool to room temperature, and then IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.7 mM was added, and expression was induced at 14°C for 20 hours to obtain the expressed bacterial liquid.

[0093] The expressed bacterial solution was centrifuged at 4000 rpm for 30 min at 4°C to collect the precipitated bacteria. At this time, the target fusion protein (Strep_8His_SUMO_Mouse SPO11) was in the bacteria. Then, the precipitated bacteria were lysed by homogenizer at 4°C and 750 bar for 3 times until the mixture of bacterial lysate was uniform and transparent. The OD value before and after the lysis of the bacteria was calculated. 600The cells were centrifuged at 30,000 g for 30 min to collect the supernatant. The lysis buffer included 1300 mM NaCl, 35 mM Tris-HCL, 40 mM imidazole, 0.7 mM PMSF, 80 μg / ml DNaseI, 7% glycerol, 0.6% TritonX-100 and 1.5 mM MgCl2.

[0094] The above supernatant was subjected to Ni affinity chromatography. A 1 mL Ni filler pre-packed column was prepared, HisTrap FF, Cytiva, catalog number 1753190. The column was first rinsed with 7 mL of water, and then equilibrated with pre-cooled 8 mL of wash buffer 1 (pH 7.5); wash buffer 1 (pH 7.5) contained 35 mM Tris-HCL, 1250 mM NaCl and 40 mM imidazole; then the Ni column was loaded for the first time, the supernatant after bacterial lysis was passed through the Ni column at a flow rate of 0.6 mL / min, and the flow-through 1 sample was collected. The target fusion protein contained an 8xHis affinity tag and a His affinity tag that could bind to the Ni filler, so the Ni filler could bind to the target fusion protein, and the protein in the flow-through was a miscellaneous protein that could not bind to the Ni filler; then the Ni column was washed for the first time with 15 mL of pre-cooled Wash 1. The column was washed with Elute buffer 1 at a flow rate of 0.6 mL / min, and the wash buffer 1 was collected for retention. The wash buffer contained 40 mM imidazole, which could wash away the impurity proteins that were non-specifically bound to or weakly bound to the Ni filler through competition, thereby increasing the purity of the target fusion protein. Then the Ni column was eluted for the first time, and the column was washed with 7 mL of pre-cooled Elute buffer 1 at a flow rate of 0.6 mL / min. The eluate 1 was collected. The eluate contained 400 mM imidazole, and the target fusion protein or other impurity proteins that were not washed away by Wash buffer 1 (pH 7.5) on the Ni filler were eluted through competition. The components of Elute buffer 1 (pH 7.5) were 35 mM Tris-HCL, 1250 mM NaCl and 400 mM imidazole.Then, the Strep affinity tag was used to further improve the purity of the fusion protein; a 1mL Strep filler pre-packed column was prepared, the column was first rinsed with 7mL water, and then the column was balanced with pre-cooled 8mL wash buffer 2, the wash buffer 2 (pH7.5) composition was 35mM Tris-HCL and 1250mM NaCl; then the Strep column (Strep column: STarm Streptactin beads, Tiandirenhe, catalog number SA092) was loaded, 1250mM sodium chloride eluent 1 was passed through the Strep column, the flow rate was 0.6mL / min, and the flow-through 2 was collected for sample retention. The target fusion protein contained a Strep II affinity tag, and the Strep II affinity tag was bound to the Strep filler, so the Strep filler could bind to the target fusion protein, and the protein in the flow-through was a miscellaneous protein that could not bind to the Strep filler; then the Strep column was washed with 15mL pre-cooled Wash buffer 2. Rinse the column at a flow rate of 0.6 mL / min, collect the wash buffer 2 sample, which can wash away the impurity proteins that are non-specifically bound to the Strep filler or weakly bound, thereby improving the purity of the target fusion protein; then elute the Strep column, rinse the column with 7 mL of pre-cooled Elute buffer 2, and collect the eluent 2, which contains 1.5 mM D-biotin. Through competition, the target fusion protein or other impurity proteins not washed away by Wash buffer 2 on the Strep filler are eluted, and the protein concentration of the eluent 2 is detected by ultraviolet spectrophotometry. The components of Elute buffer 2 (pH 7.5) are 35 mM Tris-HCL, 1250 mM NaCl and 1.5 mM D-biotin. Then, ULP1 enzyme (ULP1 enzyme: elution buffer protein = 1:100-500) was used for enzyme digestion at 4°C for 4 hours to obtain a protein solution after digestion. ULP1 enzyme can specifically recognize the SUMO tag, so that the target fusion protein (Strep_8His_SUMO_Mouse SPO11) is cut into two parts, namely the Strep_8his_SUMO part and the Mouse SPO11 target protein part. Next, a Ni column is used to bind the Strep_8his_SUMO part, ULP1 enzyme and the undigested target fusion protein to separate it from the Mouse SPO11 target protein (the Strep_8his_SUMO part, ULP1 enzyme and the undigested Strep_8His_SUMO_Mouse SPO11 fusion protein all contain a His tag and can be bound by the Ni column, while Mouse SPO11 does not contain a His tag and cannot be bound to the Ni column).Then prepare 1mL Ni filler pre-loaded column, first rinse the column with 7mL water, and then equilibrate the column with 8mL pre-cooled wash buffer 3, the composition of Wash buffer 3 (pH7.5) is 35mM Tris-HCL, 1250mM NaCl and 40mM imidazole; then load the Ni column for the second time, pass the protein after enzyme digestion through the Ni column, the flow rate is 0.6mL / min, collect the flow-through 3, the target Mouse SPO11 protein after enzyme digestion is in the flow-through 3; then elute the Ni column for the second time, rinse the column with 7mL pre-cooled Elute buffer 3, collect the eluate 3 sample, the eluted is Strep_8his_SUMO part, ULP1 enzyme and Strep_8His_SUMO_Mouse SPO11 fusion protein that is not digested by enzyme, that is, the specific domain of Mouse SPO11 protein; wherein, Elute buffer 3 (pH7.5) contains 35mM Tris-HCL, 1250mM NaCl and 400mM imidazole. Then, 12% denatured SDS PAGE was run to detect the distribution and purity of the target protein in each step of the sample. The flow-through liquid 3 was concentrated using a 5kDa ultrafiltration concentrator tube and replaced with 1250mM NaCl, pH7.4 PBS, and the sample purity was tested by SDS PAGE run. The results are as follows. Figure 7 and Figure 8 shown.

[0095] Table 1:

[0096]

[0097] Table 2:

[0098]

[0099] Figure 3 and Figure 4 The results of 1% agarose gel electrophoresis of the double enzyme digestion products are shown in Figure 1, where lanes 1, 2, and 3 are PCR products double digested with BamHI and EcoRI, lanes 5 and 6 are Alp_PET_StHis_SUMO empty vector double digested with BamHI and EcoRI, and lanes 4 and 7 are DNA Markers (Qingke Biotechnology 250bp DNA Ladder).

[0100] Figure 5 It is the double enzyme digestion vector map, that is, the expression vector Alp_PET_StHis_SUMO snapgene map (5641bp after double enzyme digestion).

[0101] Figure 6The results of colony PCR electrophoresis. Single colony 1 and single colony 3 have positive bands (1131bp). Lanes 1, 2, and 3 are the PCR products of single colonies 1, 2, and 3. Lane 4 is DNA Marker (Qingke Biotechnology DL5000Plus DNA Ladder).

[0102] Figure 7 and Figure 8 The electrophoresis results of the samples during the purification process are shown in Figure 2. Figures 7-8 It can be seen that Mouse SPO11 protein dimerization occurred during the concentration process, among which, Strep_8His_SUMO_Mouse SPO11 fusion protein: protein molecular weight 29.7kDa; Strep_8His_SUMO part: protein molecular weight 14.0kDa, SUMO protein is a ubiquitin protein, which itself will form a tertiary domain, and its apparent molecular weight will be slightly larger than the theoretical molecular weight; Mouse SPO11 target protein: protein molecular weight 15.7kDa; ULP1 enzyme: protein molecular weight 32.0kDa.

[0103] Figure 7 In the figure, lane 1 is BSA control (2 μg), lane 2 is Marker (three-color pre-stained protein Marker, Yazyme, product number WJ103), lane 3 is whole cells (before lysis), lane 4 is the supernatant of whole cell lysis and centrifugation, lane 5 is the whole cell lysis and centrifugation precipitate, lane 6 is flow-through solution 1, lane 7 is wash solution 1, lane 8 is eluent 1, lane 9 is flow-through solution 2, lane 10 is wash solution 3, lane 11 is eluent 4, lane 12 is the protein solution after enzyme cleavage, lane 13 is flow-through solution 3, and lane 14 is eluent 3.

[0104] Figure 8 In the figure, lane 1 is Mouse SPO11, and lane 2 is Marker (three-color prestained protein marker, Yazyme, catalog number WJ103).

[0105] Fig. 9The results of induced expression of SPO11 fusion protein, wherein lane 1 is MBP SPO11 strain before induction-1, lane 2 is Marker (three-color prestained protein Marker, Yazyme, catalog number WJ103), lane 3 is MBP SPO11 strain after induction-1, lane 4 is MBP SPO11 strain before induction-2, lane 5 is MBP SPO11 strain after induction-2, lane 6 is GST SPO11 strain before induction-1, lane 7 is GST SPO11 strain after induction-1, lane 8 is GST SPO11 strain before induction-2, lane 9 is GST SPO11 strain after induction-2, lane 10 is SUMO SPO11 strain before induction-1, lane 11 is SUMO SPO11 strain after induction-1, lane 12 is SUMO SPO11 strain before induction-2, lane 13 is SUMO SPO11 strain after induction -2, lane 14 is BSA control (2 μg).

[0106] Fig.10 The results of induced expression of SPO11 fusion protein, where lane 1 is whole cells (before lysis), lane 2 is the supernatant of whole cell lysis and centrifugation, lane 3 is the precipitate of whole cell lysis and centrifugation, lane 4 is the flow-through, lane 5 is wash solution 1, lane 6 is wash solution 2, lane 7 is the eluate, lane 8 is the filler sample, lane 9 is BSA control (2 μg), and lane 10 is Marker (three-color prestained protein Marker, Yazyme, product number WJ103).

[0107] Fig.11 This is the SDS PAGE detection gel image of the purification process, wherein lane 1 is BSA control (2 μg), lane 2 is Marker (three-color pre-stained protein Marker, Yazyme, catalog number WJ103), lane 3 is whole cells (before lysis), lane 4 is whole cell lysis centrifugation supernatant, lane 5 is whole cell lysis centrifugation precipitate, lane 6 is flow-through solution 1, lane 7 is wash solution 1, lane 8 is eluent 1, lane 9 is flow-through solution 2, lane 10 is wash solution 3, lane 11 is eluent 4, lane 12 is protein solution after enzyme digestion, lane 13 is flow-through solution 3, and lane 14 is eluent 3; from Fig.11 It can be seen that by comparing lanes 8 and 11, after strepII+His dual affinity tag purification, the number and proportion of impurity proteins were significantly reduced, and the purity of the target fusion protein was significantly improved; after single his tag affinity purification (lane 8), the purity of the fusion protein was 23%, and the number of impurity protein bands was 18; after strepII+His dual tag affinity purification (lane 11), the purity of the fusion protein was 39%, the purity increase ratio was 69.6%, and the number of impurity protein bands was 10.

[0108] Comparative Example 1:

[0109] The other parts are the same as Example 1, except that the fusion tags are replaced with MBP, GST and SUMO, and the expression of SPO11 obtained is as follows Fig. 9 shown.

[0110] Comparative Example 2:

[0111] The other parts are the same as Example 1, except that the NaCl concentration of all buffer systems is replaced with 500mM. The results are as follows Fig.10 shown.

[0112] From the comparison between Example 1 and Comparative Example 1, it can be seen that when MBP, GST, and SUMO fusion tags are used, the expression levels of MBP-SPO11 and GST-SPO11 are extremely low or non-existent.

[0113] From the comparison between Example 1 and Comparative Example 2, it can be seen that when the salt concentration in the purification buffer system is ≤500mM, the SDS PAGE results of the purification process indicate that the target fusion protein is not dissolved in the supernatant of the broken bacteria, but in the precipitate of the broken bacteria, and it is extremely difficult to extract the protein in the precipitate of the broken bacteria. After the salt concentration in the purification buffer system is increased to 1000-1500mM, the target fusion protein is in the supernatant of the broken bacteria, which is convenient for subsequent steps such as affinity chromatography.

[0114] The present invention adds a SUMO solubility-promoting tag when expressing Mouse SPO11, thereby solving the problem of the target protein forming inclusion bodies in the large intestine expression system, and the SUMO tag increases the expression amount of the soluble target protein in the Escherichia coli expression system, helps the protein to fold correctly, and improves the protein stability; the present invention uses a high salt system, 1000-1500mM NaCl, in the purification process, to improve the stability and solubility of the Mouse SPO11 protein, and to improve the yield and purity of Mouse SPO11; the present invention designs a Strep II and his dual affinity tag, and removes non-target proteins as much as possible through two affinity chromatography, thereby being more conducive to obtaining a high-purity Mouse SPO11 protein; the ULP1 enzyme of the present invention can specifically recognize the SUMO tag, and the ULP1 enzyme has extremely high enzymatic cleavage efficiency, and can complete 95% fusion protein cleavage in a short time, thereby being more conducive to obtaining a high-purity target protein.

[0115] In the technical solution provided in this article, the detailed information of the relevant reagents and biological enzymes used are as follows:

[0116] Prime STAR MAX DNA Ploymerase is from Takara, catalog number R045; SanPrep column DNA gel recovery kit, Sangon Biotechnology, catalog number B518131; BamHI-HF, NEB, catalog number R3136; EcoRI-HF, NEB, catalog number R3101; SanPrep column DNA gel recovery kit, Sangon Biotechnology, catalog number B518131; T4 ligase, NEB, catalog number M0202; Easytaq DNA Polymerase high-efficiency amplification DNA polymerase and EasyTaq buffer set (Beijing Quanshijin, catalog number AP111); High Pure dNTPs (2.5mM) (Beijing Quanshijin, catalog number AD101); ULP1 enzyme: Wozhen Biotechnology (Foshan) Co., Ltd.

[0117] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0118] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for expressing a specific domain of Mouse SPO11 protein, characterized in that: It includes the following steps: Using the Mouse SPO11 protein sequence as a template, primers are designed for PCR amplification to obtain the Mouse SPO11 gene fragment after PCR amplification; In the Alp_PET_StHis_SUMO empty vector, Strep tag II affinity tag, 8x his affinity tag and SUMO solubility tag were designed in sequence at the N-terminus of the multiple cloning site to obtain the expression vector Alp_PET_StHis_SUMO; The Mouse SPO11 gene fragment amplified by PCR is subjected to gel electrophoresis, and then the gel is cut and recovered, and then the fragment is double-digested with BamHI and EcoRI at the same time as the expression vector Alp_PET_StHis_SUMO to obtain a double-digested fragment and a double-digested vector; The double-enzyme-digested fragment and the double-enzyme-digested vector are excised and recovered by gel excision, and then ligated using T4 ligase to obtain a ligation product, wherein the molar amount of the double-enzyme-digested fragment and the molar amount of the double-enzyme-digested vector is 4-6:1; All the ligation products were transformed into DH5α competent medium, and after transformation, all the transformation products were spread on Kana resistance LB solid plates with a final concentration of 30-70 μg / mL to obtain ligation single colonies; The connected single colony is subjected to colony PCR, and then inoculated into a Kana resistance LB liquid culture medium with a concentration of 30-70 μg / mL for colony culture to obtain a PCR bacterial solution, and the PCR bacterial solution is subjected to plasmid extraction and sequencing, and a successfully constructed plasmid is obtained when the result is correct; Inducing the expression of the plasmid to obtain an expressed bacterial solution; Purifying the expressed bacterial solution to obtain the specific structural domain of Mouse SPO11 protein; The purification comprises the following steps: The expressed bacterial solution is centrifuged at 3500-4500 rpm at 3-5°C, the precipitated bacterial bodies are collected, and then the precipitated bacterial bodies are crushed and lysed, and the supernatant is obtained by centrifugation after lysis, and the supernatant is subjected to Ni affinity chromatography, and then Strep affinity chromatography, and then ULP1 enzyme is used for enzyme digestion, and then Ni affinity chromatography is performed to obtain the specific domain of the Mouse SPO11 protein; The concentration of NaCl in the purification buffer system of the Ni affinity chromatography and the Strep affinity chromatography is 1000-1500 mM; The amino acid sequence of the Mouse SPO11 protein is: AGATRVASSSEVLTAIENIIQDIIKSLARNEVPAFTIDNRSSWENIMFDDSVGLRMIPQCTTRKIRSDSPKSVKKFALILKVLSMIYKLIQSDTYATKRDIYYTDSQLFGNQAAVDSAIDDISCMLKVPRRSLHVLST; The primers for PCR amplification include a forward primer and a reverse primer; the forward primer is: 5'-GTCACCACGCGGATCCGCTGGTGCAACCCGTGTGGCAAG-3', and the reverse primer is: 5'-CCATCGACCGGAATTCTTAGGTACTCAGCACATGCAGGCTACGAC-3'; The conditions of the colony PCR were as follows: forward primer: 5'-ATGCCGGCCACGATGCGTC-3', reverse primer: 5'-CCGGATATAGTTCCTCCTTTC-3'; The fragment after double enzyme digestion is GGATCCGCTGGTGCAACCCGTGTGGCAAGCAGCAGTGAAGTGCTGACCGCAATTGAAAATATTATCCAGGATATCATCAAGAGCCTGGCACGCAATGAAGTTCCGGCATTCACTATTGATAACCGCAGCAGCTGGGAAAATATTATGTTTGATGACAGCGTGGGCCTGCGTATGATTCCGCAGTGTACCACCCGTAAAATTCGCAGCGAT AGTCCGAAAAGCGTGAAAAAAATTTGCACTGATTCTGAAGGTGCTGAGTATGATCTATAAGCTGATTCAGAGTGACACCTATGCAACCAAACGTGATATCTATTACACCGATAGCCAGCTGTTTGGCAATCAGGCAGCAGTTGATAGTGCAATTGATGATATTAGCTGCATGCTGAAAGTGCCGCGTCGTAGCCTGCATGTGCTGAGTACCTAAGAATTC; The Ni affinity chromatography is a His affinity tag; The Strep affinity chromatography is a Strep II affinity tag; The cracking conditions are 3-5°C and 600-900bar; The lysis uses a lysis buffer, which includes 1000-1500 mM NaCl, 20-50 mM Tris-HCL, 20-60 mM imidazole, 0.5-1.0 mM PMSF, 50-100 μg / ml DNaseI, 5-10% glycerol, 0.1-1.0% TritonX-100 and 1-2 mM MgCl2.

2. The method according to claim 1, characterized in that The inducing expression comprises the following steps: The plasmid was transformed into the competent E. coli expression strain CODON PLUS, and then the transformation product was spread on a KANA resistance LB solid plate with a final concentration of 30-70 μg / mL, and then cultured at 35-39° C. for 16-24 hours to obtain a single plasmid colony; Take a single colony of the plasmid and inoculate it in a KANA resistance LB liquid medium with a final concentration of 30-70 μg / mL, then culture it at 35-39°C for 16-24 h, and then expand the culture at 35-39°C until the OD 600 =0.6~0.8, then placed in tap water to cool to room temperature, then added with isopropyl-β-D-thiogalactoside at a final concentration of 0.5~1.0 mM, induced expression at 12~16°C for 16~24h, to obtain the expressed bacterial solution.

3. The method according to claim 1, characterized in that The conditions of the gel electrophoresis are: 0.5-1.5% agarose, 25-35 min; The double enzyme digestion conditions are 35-39°C, 16-24h; The connection conditions are 15-17° C., 4-6 h; The conditions for the colony culture are 35-39° C., 16-24 h, and 210-230 rpm.

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