Expression vector of recombinant BrCas12b protease, recombinant engineering bacterium and application of recombinant BrCas12b protease

Abstract

The invention provides an expression vector of a recombinant BrCas12b protein, a recombinant engineering bacterium, a preparation method of the recombinant BrCas12b protein and application of the recombinant BrCas12b protein. The method comprises the following steps: expressing the recombinant BrCas12b protein with a His tag through bacteria to obtain a supernatant of the BrCas12b protein, and purifying the supernatant through a Ni-NTA chromatographic column to obtain the purified recombinant BrCas12b protein. The invention provides a simple, convenient and efficient Brcas12b recombinant protein expression and purification method, and solves the problems of low yield, low purity and the like in the existing method.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to gene editing technology, and in particular to a method for purifying BrCas12b protein and an application thereof. Background Art

[0002] BrCas12b is an emerging CRISPR-Cas system nuclease that has attracted widespread attention in the field of gene editing and molecular diagnosis in recent years. The CRISPR-Cas system is a natural immune mechanism used by bacteria and archaea to resist the invasion of foreign genomic materials (such as viral DNA). The system consists of CRISPR sequences and Cas proteins, of which Cas proteins are responsible for cutting foreign DNA and protecting cells from infection. With the widespread application of CRISPR-Cas technology in gene editing, various types of Cas proteins have been discovered and studied one after another, among which BrCas12b has received special attention due to its unique characteristics.

[0003] BrCas12b was originally isolated from thermophilic bacteria and has higher thermal stability than other Cas12 family members (such as Cas12a and Cas12c). This feature enables BrCas12b to maintain its nuclease activity under high temperature conditions, making it suitable for use in environments that require high temperature reactions. In addition, BrCas12b recognizes a wider range of PAM (Protospacer Adjacent Motif) sequences, usually 5'-TTN or other similar sequences, which enables BrCas12b to target more diverse genomic regions during gene editing.

[0004] BrCas12b initiates the cleavage reaction by recognizing and binding crRNA (CRISPR RNA) to pair with the target DNA sequence. When targeting the DNA sequence, BrCas12b can produce DNA breaks with 3' overhangs downstream of the PAM sequence. This cleavage mechanism has broad application prospects in gene editing and gene modification, especially in gene editing tasks that require high precision and high specificity.

[0005] In addition, BrCas12b has been used to develop rapid and sensitive nucleic acid detection methods due to its stability at high temperatures, especially in the field of molecular diagnosis. For example, in real-time detection of pathogens or screening of gene mutations, BrCas12b has become an ideal candidate for the development of new diagnostic tools due to its high sensitivity and specificity. However, the current existing technologies have problems such as low yield and low purity of BrCas12b. Summary of the invention

[0006] The object of the present invention is to provide a method for purifying recombinant BrCas12b protein. The method is simple in process, the obtained target protein has high purity and high cleavage activity. The present invention provides an efficient recombinant BrCas12b protein expression and purification method, comprising the following steps:

[0007] An expression vector for expressing a recombinant BrCas12b protein, comprising a backbone vector with a HIS tag, and a coding gene for the BrCas12b protein inserted into the backbone vector; the nucleotide sequence of the coding gene for the BrCas12b protein is SEQ ID NO: 1.

[0008] In one embodiment according to the present invention, the backbone vector is selected from PET 28a and PGEX-6P; preferably pET28a(+).

[0009] Another aspect of the present invention provides a recombinant engineered bacterium for expressing a recombinant BrCas12b protein, comprising a host bacterium and the above-mentioned expression vector.

[0010] In one embodiment of the present invention, the host bacteria is Escherichia coli; preferably, the host bacteria is Origami 2 (DE3), Rosetta 2 (DE3), BL21 (DE3), BL21 (DE3)

[0011] PlysS, Transetta (DE3) or Tuner (DE3); preferably Rosetta 2 (DE3).

[0012] The present invention further provides the use of the above-mentioned expression vector or recombinant engineered bacteria in preparing recombinant BrCas12b protein.

[0013] Another aspect of the present invention provides a method for preparing a recombinant BrCas12b protein, comprising:

[0014] 1) Introducing the above expression vector into a host bacterium to induce expression; or inducing expression in the above recombinant engineered bacterium;

[0015] 2) Breaking the bacteria and isolating the recombinant BrCas12b protein;

[0016] 3) Purification by Ni-NTA chromatography column to obtain recombinant BrCas12b protein.

[0017] In step 1) according to one embodiment of the present invention, the expression vector is a recombinant vector composed of a coding gene with a nucleotide sequence of SEQ ID NO: 1 connected to an expression vector plasmid pET28a(+); preferably, the host bacteria is Rosetta2(DE3).

[0018] In one embodiment according to the present invention, step 2) is achieved by high pressure sterilization centrifugation comprising the following steps:

[0019] The BrCas12b bacteria collected in step 1) are mixed and suspended with a balancing solution, pre-cooled and then high-pressure broken, centrifuged at high speed, and the supernatant is collected to obtain a protein solution; the balancing solution contains 50mM HEPES, 100mM KCl, 10% glycerol, PH7.5.

[0020] In one embodiment of the present invention, the balance solution and the bacteria are resuspended in a weight: volume ratio of 1:10; 700-800 MPa high pressure is used; 4°C, 12000 rpm high-speed centrifugation for 15 minutes is used to obtain the ruptured supernatant.

[0021] In one embodiment according to the present invention, the chromatography column purification is achieved by a method comprising the following steps:

[0022] The Ni-NTA chromatography column was alternately rinsed with pure water and equilibration solution for 5-10 column volumes, the protein solution collected in step 2) was added, and the purified protein was collected and eluted with an eluent containing imidazole; the eluent contained 50 mM HEPES, 100 mM KCl, 20-500 mM imidazole, 10% glycerol, pH 7.5;

[0023] Preferably, the Ni-NTA chromatography purification filler is HisSep Ni-NTA Agarose Resin; the filler dosage is 100 mL per 100 g wet weight of broken bacteria, and the incubation conditions are: 4° C., 2 h.

[0024] The beneficial effects of the above technical solution of the present invention are as follows:

[0025] The present invention provides a simple and efficient method for expressing and purifying the Brcas12b recombinant protein, which overcomes the problems of low yield and low purity in the existing methods and has important scientific research and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the result of protein expression, purification and identification of pET28a(+)-Brcas12b;

[0027] Figure 2 This is the result of identifying the activity of Brcas12b protein. DETAILED DESCRIPTION

[0028] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0029] Unless otherwise specified, all reagents used in this example were of analytical grade, and the progress of all chemical reactions was detected by thin layer chromatography.

[0030] The present invention cloned and expressed the Brcas12b gene, constructed it into a pET28a(+) plasmid, induced its recombinant expression in Rosetta2(DE3) under 1mM IPTG and 37°C conditions, and then lysed the bacteria with high pressure and centrifuged, and purified the prepared recombinant protein through a Ni-NTA chromatography column.

[0031] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0032] Materials and reagents

[0033] 1. Plasmid

[0034] The pET28a(+) plasmid was purchased from Jinkairui.

[0035] 2. Main Reagents

[0036] Kanamycin Sulfate Maclean Chloramphenicol Maclean IPTG (isopropyl-β-D-thiogalactopyranoside) Biosharp 12% PAGE Color Gel Quick Preparation Kit Biosharp <![CDATA[Capto TM S ImpAct Ion Column]]> Cytiva His-Sep Ni-NTA Agaroses Resin Yeasen

[0037] 3. Main instruments and equipment

[0038] JFZD-200B Oscillator Hangzhou Jingpei Instrument Technology Co., Ltd. High-speed refrigerated centrifuge Lu Xiangyi Ultrasonic cell disruptor Ningbo Xinzhi Biotechnology Co., Ltd. Chromatography AKTA pure150 Full wavelength microplate reader Thermo Scientific Imager Tanon High pressure homogenizer Ningbo Xinzhi Biotechnology Co., Ltd.

[0039] Example 1 Cloning and construction of Brcas12b gene

[0040] 1) Construction of pET28a(+)-Brcas12b vector

[0041] A Brcas12b CDS sequence with a length of 3305 bp was designed, and its nucleotide sequence was shown in SEQ ID NO.1. The above sequence was synthesized by whole gene synthesis and cloned into a pET28a(+) plasmid to construct a pET28a(+)-Brcas12b vector. The expression of the pET28a(+)-Brcas12b fusion protein used the inherent T7 promoter, lac operator, RBS and T7 terminator on the pET28a(+) vector, and the protein expression could be regulated by isopropyl-β-D-thiogalactopyranoside (IPTG).

[0042] 2) The recombinant plasmid was transformed into the expression strain Rosetta2 (DE3) to obtain the recombinant Escherichia coli pET28a (+) / Brcas12b / Rosetta2 (DE3) that can express Brcas12b.

[0043] The nucleotide sequence of the gene encoding the Brcas12b protein is shown in SEQ ID NO: 1:

[0044]

[0045]

[0046]

[0047] Example 2 Inducible expression of Brcas12b protein

[0048] 1) Activation of bacteria: The candidate protein expression bacteria pET28a(+) / Brcas12b / Rosetta2(DE3) stored at -80°C were inoculated at a ratio of 1:1000 for activation, i.e., 10 μL of glycerol bacterial solution was added to 10 mL of LB medium containing Kana and CHL resistance, and cultured overnight at 37°C, 200 rpm for 16 h.

[0049] 2) Secondary activation induction: Inoculate bacteria at a ratio of 1:100 for secondary activation. Take 200 μL of overnight bacterial solution and add it to 20 mL of LB medium containing Kana and CHL resistance. Incubate at 37°C for 4 h at 180 rpm. Secondary activation until OD 600 When the pH value is 0.6, add IPTG to a final concentration of 1 mM, place in a shaker at 37°C, 180 rpm for 3 h. (Preparation of IPTG solution: dissolve 2.38 g IPTG in 10 mL Grade I water, filter with a 0.22 μm sterile filter after complete dissolution).

[0050] 3) Take out the induced expression bacterial solution, centrifuge at 4°C, 6000 rpm for 15 min, discard the supernatant, add 1-2 mL of balancing solution (50 mM HEPES, 100 mM KCl, 10% glycerol, pH 7.5), resuspend and mix, ultrasonically disrupt the bacteria for 15 min, collect the disrupted solution, centrifuge at 4°C, 12000 rpm for 15 min, and separate the supernatant and precipitate.

[0051] 4) Process the supernatant and precipitate: take 20 μL of supernatant and add 5 μL of 5× protein loading buffer, boil for 5 min, and then centrifuge at 14000 rpm for 3 min; add 30 μL of equilibration solution to the precipitate to resuspend the bacteria, take 20 μL of the resuspended bacteria solution and add 5 μL of 5× protein loading buffer, boil for 5 min, and then centrifuge at 14000 rpm for 3 min.

[0052] 5) Pour 12% concentrated gel into the gel plate, add distilled water to flatten the gel, place it at room temperature for 30 minutes to solidify, drain the distilled water on the upper layer, and then pour in 12% separation gel, immediately insert the comb, and place it at room temperature for 30 minutes to solidify for use. Take 10 μL of the treated supernatant and precipitate, respectively, and perform SDS-PAGE electrophoresis. The voltage is first 80V for 30 minutes, then adjusted to 180V. After electrophoresis for 1-2 hours, take out the gel, place it in the instant blue staining solution for shaking and staining, and then place it in water for shaking and decolorization. Observe the results under the imaging system. Brcas12b is a soluble protein.

[0053] Example 3 Preparation of Brcas12b recombinant protein

[0054] 1) Amplification and culture to obtain protein: 400 μL of pET28a(+) / Brcas12b / Rosetta2(DE3) bacterial solution stored in a 4°C refrigerator was added to 20 mL of LB medium containing Kana and CHL resistance for primary activation, and cultured at 200 rpm and 37°C for 4 hours. Then, 20 mL of the primary activated bacterial solution was added to 2000 mL of LB medium containing Kana and CHL resistance for secondary activation, and cultured at 37°C for 4 hours. 2 mL of IPTG (mother solution concentration is 1 M) was added and induced at 180 rpm in a 37°C shaker for 3 hours, and then centrifuged at 4°C and 12000 rpm for 15 minutes to collect the bacteria. 50 mL of balancing solution was added to resuspend the bacteria, and the bacterial solution was high-pressure lysed (pressure was 700-800 mpa), and the lysed solution was collected.

[0055] 2) Binding of protein to filler: After breaking the bacteria, the solution was centrifuged at 12000rpm, 50min, and 4℃. The supernatant was added to the Ni-NTA chromatography column and placed on a 4℃ rotary shaker for 2h. The penetration liquid was collected and sampled. The impurities were washed with 5 column volumes of 20mM imidazole and 5 column volumes of 50mM imidazole, and samples were taken. The Brcas12b protein was eluted with 5 column volumes of 100mM imidazole-500mM imidazole, and samples were taken for SDS-PAGE electrophoresis. Finally, the filler was washed with 500mM imidazole, rinsed with pure water, and stored in 20% ethanol.

[0056] 3) Protein concentration and buffer replacement: The eluted protein solution was transferred to a dialysis bag with a molecular weight of 14,000. Place the dialysis bag in 1L of concentration buffer (ingredients include 500mM NaCl, 20mM sodium acetate, 0.1mM EDTA, 0.1mM DTT, 0.5mM PMSF, 5% PEG, 10% glycerol) and stir overnight at 4°C. The next day, transfer the protein to 1L storage buffer (ingredients include 50mM NaCl, 20mM sodium acetate, 0.1mM EDTA, 0.1mM DTT, 0.5mM PMSF, 50% glycerol) and stir overnight at 4°C. The next day, measure the protein concentration. Finally, 15mg of HuCas12a protein was obtained per liter of bacterial solution, with a purity of more than 90%.

[0057] 4) Perform 12% SDS-PAGE electrophoresis, stain with instant blue dye for 15 minutes, and place in water for oscillation to decolorize. The results of gel scanning imaging system observation showed that the molecular weight of Brcas12b protein was 140 kDa, which was consistent with the expected protein molecular weight (results as shown in Figure 1 shown).

[0058] 5) Protein activity assay: While BrCas12b recognizes and cuts the target double-stranded DNA (reaction substrate), its high-efficiency cutting activity of single-stranded DNA (ssDNA) of any sequence is activated. By designing probes with fluorescent markers or other small molecule markers at both ends, the non-specific activity of CRISPR / Cas12b on ssDNA cutting is used to achieve signal amplification, and the cutting activity of BrCas12b recombinant protein is measured by real-time fluorescence method.

[0059] Reaction system:

[0060] Ingredient name Reaction volume / final concentration 10×Reaction Buffer 1 1× Brcas12b 2μL gRNA 50nmol Fluorescent probes 40nM <![CDATA[H 2 The]]> Add the above to 48 μL Reaction substrate 2μL

[0061] The results of fluorescence detection showed that the strong positive reaction curve reached the plateau in 10-15 minutes. Figure 2 shown).

[0062] The above is 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An expression vector for expressing recombinant BrCas12b protein, characterized in that, It comprises a backbone vector with a HIS tag, and a gene encoding a BrCas12b protein inserted into the backbone vector; the nucleotide sequence of the gene encoding the BrCas12b protein is SEQ ID NO:

1.

2. The expression vector according to claim 1, characterized in that The skeleton carrier is selected from PET 28a and PGEX-6P; preferably pET28a(+).

3. A recombinant engineered bacterium for expressing recombinant BrCas12b protein, characterized in that, It comprises a host bacterium and the expression vector as claimed in claim 1 or 2.

4. The recombinant engineered bacterium according to claim 3, characterized in that The host bacteria is Escherichia coli; preferably, the host bacteria is Origami 2 (DE3), Rosetta 2 (DE3), BL21 (DE3), BL21 (DE3) PlysS, Transetta (DE3) or Tuner (DE3); preferably Rosetta 2 (DE3).

5. the purposes of the expression vector as claimed in claim 1 or 2 or the recombinant engineering bacteria as claimed in claim 3 or 4 in preparing recombinant BrCas12b protein.

6. A method for preparing a recombinant BrCas12b protein, characterized in that, include: 1) Introducing the expression vector as claimed in claim 1 or 2 into a host bacterium to induce expression; or inducing expression of the recombinant engineered bacterium as claimed in claim 3 or 4; 2) Breaking the bacteria and isolating the recombinant BrCas12b protein; 3) Purification by Ni-NTA chromatography column to obtain recombinant BrCas12b protein.

7. The preparation method according to claim 6, characterized in that: In step 1), the expression vector is a recombinant vector composed of a coding gene with a nucleotide sequence of SEQ ID NO: 1 connected to an expression vector plasmid pET28a(+); preferably, the host bacteria is Rosetta2(DE3).

8. The preparation method according to claim 6 or 7, characterized in that: Step 2) is achieved by high pressure sterilization centrifugation comprising the following steps: The BrCas12b bacteria collected in step 1) are mixed and suspended with a balancing solution, pre-cooled and then high-pressure broken, centrifuged at high speed, and the supernatant is collected to obtain a protein solution; the balancing solution contains 50mM HEPES, 100mM KCl, 10% glycerol, PH7.

5.

9. The preparation method according to claim 8, characterized in that: The balance solution and the bacteria were resuspended in a weight: volume ratio of 1:10; high pressure of 700-800 MPa was used; and high-speed centrifugation at 4°C and 12000 rpm for 15 minutes was used to obtain the supernatant for rupturing the bacteria.

10. The preparation method according to any one of claims 6 to 9, characterized in that: The chromatography column purification is achieved by a method comprising the following steps: The Ni-NTA chromatography column was alternately rinsed with pure water and equilibration solution for 5-10 column volumes, the protein solution collected in step 2) was added, and the purified protein was collected and eluted with an eluent containing imidazole; the eluent contained 50 mM HEPES, 100 mM KCl, 20-500 mM imidazole, 10% glycerol, pH 7.5; Preferably, the Ni-NTA chromatography purification filler is HisSep Ni-NTA Agarose Resin; the filler dosage is 100 mL per 100 g wet weight of broken bacteria, and the incubation conditions are: 4° C., 2 h.

Images