CN111893105A

CN111893105A - Cas9 protein expression and purification method

Info

Publication number: CN111893105A
Application number: CN202010766185.2A
Authority: CN
Inventors: 吴正红; 潘秀华; 祁小乐
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-08-03
Filing date: 2020-08-03
Publication date: 2020-11-06

Abstract

The invention discloses an expression and purification method of Cas9 protein, and belongs to the technical field of biotechnology and genetic engineering. The Cas9 protein foreign plasmid with His6 label is selected to be expressed in a large amount in an escherichia coli expression system, and finally Ni is adopted²⁺And purifying the metal chelating resin, and effectively producing the Cas9 protein in batches. The colibacillus expression system adopted by the invention has the characteristics of quick propagation, low cost, high expression quantity, easy purification of an expression product and good stability, and Ni²⁺The purification method of the metal chelating resin is convenient and rapid, the affinity and purity of the target protein are high, the obtained Cas9 protein has high purity and is correctly folded to be beneficial to forming a ribose protein compound gene medicine, so that the gene editing is finally exertedAnd (4) performance.

Description

Cas9 protein expression and purification method

Technical Field

The invention belongs to the technical field of biotechnology and genetic engineering, and particularly relates to an expression and purification method of Cas9 protein.

Background

In recent years, gene therapy has been one of the most revolutionary medical techniques with the development of DNA recombination technology and gene cloning technology, and has attracted much attention. Gene editing, one of the important means for gene therapy, is a genetic engineering technique that can precisely modify a specific target gene in the genome of an organism. The CRISPR/Cas9 system is a third generation gene editing technology that appears beyond zinc finger endonuclease (ZFN), transcription activator-like effector nucleases (TALENs), and its mediated gene editing can be used to generate transgenic models, regulate transcription, regulate epigenetics, and the like. The CRISPR/Cas9 system mainly comprises a Cas9 protein and a single-stranded guide RNA (sgRNA), the Cas9 protein causes DNA double-strand break under the guide action of the sgRNA, and a riboprotein complex formed by the two has gene editing performance, so the Cas9 protein is one of important component elements of the gene editing drug.

Currently, Cas9 protein is prone to have high endotoxin content, formation of insoluble protein in inclusion body form, incorrect folding of protein, low yield and other problems during purification, which limits the mass production of protein and affects the inherent activity of protein.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an expression and purification method of Cas9 protein, which comprises the steps of transforming exogenous Cas9 protein coding plasmid into competent escherichia coli, expressing by using an escherichia coli expression system and purifying by using metal chelating resin to prepare the Cas9 protein with gene editing performance.

In order to achieve the above object, the present invention adopts the following technical means:

a method for expression purification of Cas9 protein, comprising the steps of:

step 1, transferring pET-28b-Cas9-His6 plasmid into a host cell E.coli Rosetta (DE3) pLysS to obtain a recombinant strain Rosetta (DE3) pLysS;

step 2, transferring and activating the recombinant strain, inducing the recombinant strain to express Cas9 protein by IPTG, and centrifugally collecting bacteria by bacterial liquid to obtain escherichia coli bacteria containing Cas9 protein;

step 3, resuspending the obtained thalli by using a buffer solution, carrying out ultrasonic bacteria breaking, and collecting a supernatant;

step 4, passing the collected supernatant through a Ni-NTA resin column, washing the supernatant with a washing solution, and eluting protein with an eluent to obtain a Cas9 protein solution;

and 5, carrying out ultrafiltration concentration on the obtained Cas9 protein solution to obtain the Cas9 protein.

Further, the recombinant strain was transferred to 5mL Kan in step 2⁺&CHL⁺Culturing the culture medium at 37 ℃ and 220rpm for 16h for activation, measuring activated bacteria liquid according to 1 percent of inoculation amount after activation, and inoculating the activated bacteria liquid to Kan⁺&CHL⁺The medium was cultured at 37 ℃ and 220rpm for 2h30min, and then 0.1mM IPTG was added to induce expression.

Further, the temperature for inducing expression was 20 ℃ and the time was 8 hours.

Further, the conditions of ultrasonic bacteria breaking in the step 3 are that the ultrasonic is started for 1s and stopped for 1s, and the ultrasonic treatment is carried out for 15min in ice bath under the power of 500W.

Further, the eluent in step 4 was 300mM imidazole.

Further, ultrafiltration concentration was performed using a 50kDa ultrafiltration tube in step 5.

The expression system of the existing protein comprises escherichia coli, yeast, insects, baculovirus, mammals and the like, wherein the escherichia coli has the advantages of clear genetic background, low cost, high expression quantity, relatively simple separation and purification of an expression product and the like, and becomes the main system for expressing the existing protein. Ni²⁺The metal chelating resin protein purification method has the advantages of strong specific binding capacity, high protein purity, simple and convenient operation and the like, and can be specifically bound with the protein with the histidine tag so as to achieve the purpose of purifying the protein. Common tags of the metal chelating resin are His6, Flag, MBP, GST, HA, luciferase and the like, wherein the His6 protein tag is different from other macromolecular tags, and the small molecular weight does not influence the protein function generally.

The invention selects a Cas9 protein exogenous plasmid with His6 label to express in a large amount in an escherichia coli expression system, and finally adopts Ni²⁺And purifying the metal chelating resin, and effectively producing the Cas9 protein in batches. Meanwhile, the solubility, expression conditions, purification conditions, bacteria breaking conditions, storage conditions and the like of the exogenous Cas9 protein plasmid in an escherichia coli expression systemThe final purity, yield and gene editing activity of the Cas9 protein are responded, the Cas9 protein which is high in purity and correctly folded is obtained by screening the expression and purification conditions of the Cas9 protein engineering bacteria, and the formation of a ribose protein compound gene medicine is facilitated, so that the gene editing performance is finally exerted.

Drawings

FIG. 1 is a map of pET-28b-Cas9-His6 plasmid.

FIG. 2 shows the SDS-PAGE analysis result of the Cas9 protein engineering bacteria in example 1.

FIG. 3 shows the SDS-PAGE results of example 2 at different induction temperatures, where a is 20 ℃ and b is 37 ℃.

FIG. 4 shows the SDS-PAGE results of the Cas9 engineering bacteria induction time screening in example 2.

Fig. 5 shows the bacteria-breaking power screening result of the Cas9 protein bacterial liquid in example 2.

FIG. 6 shows the results of screening the concentrations of the heteroauximab eluents in example 2.

Fig. 7 shows the analysis result of the Ni-NTA resin column purification process of Cas9 protein in example 2.

Figure 8 is the SDS-PAGE result after concentration of Cas9 protein in example 2.

Fig. 9 shows the CD spectrometry result of Cas9 protein in example 2.

Fig. 10 shows the result of particle size measurement of Cas9 protein in example 2.

Figure 11 is the cleavage activity results of Cas9-sgRNA in example 2.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to specific examples. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.

The plasmid pET-28b-Cas9-His6 (shown in figure 1) used in the invention is purchased from Wuhan vast Ling Biotech Co., Ltd, and other reagents are all commercially available products. The experimental procedures, which are not described in detail, were performed according to the laboratory manual, e.g., molecular cloning.

Example 1

Construction of Cas9 protein engineering bacteria

Dissolving pET-28b-Cas9-His6 plasmid dry powder by 40 mu L TE Buffer, and carrying out vigorous vortex microcentrifugation; after the concentration measurement, the solution was diluted to a concentration of 10 ng/. mu.L with TE Buffer for use. Coli Rosetta (DE3) pLysS chemically competent cells were thawed on ice; add 1.5. mu.L 10 ng/. mu.L plasmid solution to every 100. mu.L competent cell, flick evenly, incubate for 30min on ice; placing the centrifugal tube in a water bath kettle at 42 ℃ for heat shock for 90s, quickly transferring the centrifugal tube to an ice water bath for standing for 2min without shaking; adding 900 μ L LB culture medium into the centrifuge tube, incubating for 45min and rejuvenating in a constant temperature shaking incubator with 37 ℃ and 150 rpm; 200 μ L of the conversion product was taken and spread evenly on Kan using a sterile spreading bar⁺And CHL⁺The solid medium of (4) was plated and cultured overnight at 37 ℃. Adding Kan and CHL with final concentrations of 30 mu g/mL and 34 mu g/mL respectively into 100mL of LB culture medium, and shaking gently to mix uniformly; larger, rounder single colonies were picked from overnight-cultured plates and inoculated into 5mL Kan⁺And CHL⁺LB culture medium; incubated at 37 ℃ for about 8 hours at 220 rpm. And (3) taking 1.5mL of sterilized EP tube, respectively adding 200 mu L of 80% sterilized glycerol and 800 mu L of turbid bacterial liquid, blowing, uniformly mixing, marking and sealing by using a sealing film to obtain the positive engineering bacteria converted with the pET-28b-Cas9 recombinant plasmid, and freezing and storing at-80 ℃ for a long time.

Three batches of strains which can be preserved for a long time are prepared by picking three times of positive monoclonals of Rosetta (DE3) pLysS positive engineering bacteria which are constructed by a heat shock method and can express exogenous plasmid pET-28b-Cas9-His 6. The supernatant after the cell wall is destroyed by ultrasonic wave is analyzed by SDS-PAGE, the result is shown in figure 2, and the three batches of engineering bacteria successfully express Cas9 protein. The theoretical value of the Cas9 protein was 160.0kDa, and the position of the band coincided with the theoretical value. Compared with the Rosetta (DE3) pLysS empty bacteria which are not transformed with the exogenous plasmid, the total bacteria and the supernatant of the transformed three batches of bacteria have obvious bands at 160.0kDa after centrifugation, and the empty bacteria have no bands, which indicates that the Cas9 protein positive engineering bacteria are successfully constructed.

Example 2

Expression and purification of Cas9 protein

1. Screening for inducible expression temperature

Taking a sterilized empty test tube, and picking the frozen pET-28b-Cas9 positive engineering bacteria are transferred to 5mL Kan⁺&CHL⁺LB medium, 37 ℃ 220rpm overnight culture for about 16 h. Transferring 50 mu L of the activated engineering bacteria into 5mL Kan according to 1% inoculation amount⁺&CHL⁺LB medium in tubes, 37 ℃ 220rpm for about 3 h. mu.L of IPTG was added to each flask of the medium to a final concentration of 0.1mM, and the resulting mixture was subjected to induction culture at 20 ℃ and 37 ℃ for 8 hours at 220 rpm. Taking out 1.5mL of the bacterial liquid from the two test tubes to an EP tube respectively, and putting the tube into an ultrasonic instrument for ultrasonic bacteria breaking for 4 h. After the disruption, 10. mu.L of each of the turbid bacterial solution and the supernatant was taken out to prepare SDS-PAGE samples, which were then analyzed by SDS-PAGE.

Escherichia coli expresses exogenous soluble recombinant plasmids, and the optimal temperature of the recombinant plasmids is the most important condition for expressing Cas9 protein by positive engineering bacteria. If the temperature is proper, the protein formed by the correct assembly and folding of the protein is in a soluble form; if the temperature is too high or too low, the expressed protein is insoluble protein aggregate and becomes inclusion body protein, and after bacteria are broken, a soluble solution cannot be obtained by centrifugation, but the bacteria are remained in the precipitate. As the growth temperature of common escherichia coli is 37 ℃ and the growth temperature of low-temperature expression bacteria is about 20 ℃, SDS-PAGE analysis is carried out on the expression conditions of the Cas9 protein at the two temperatures, and the result is shown in figure 3, a large number of bands appear in 160kDa position in the supernatant after the bacteria are broken at the temperature of 20 ℃, which indicates that the Cas9 protein expressed at the temperature can be dissolved in the supernatant, and no band appears in the supernatant after the bacteria are broken in the total bacteria at the temperature of 37 ℃, which indicates that the protein exists in an insoluble form, namely inclusion body protein, so that the temperature of 20 ℃ is a proper induced expression temperature.

2. Screening for Induction of expression initiation time

Taking a sterilized empty test tube, picking the frozen pET-28b-Cas9 positive engineering bacteria, transferring to 5mL Kan⁺&CHL⁺The medium was incubated at 37 ℃ and 220rpm overnight for about 16 hours. A200 mL bottle of Kan was prepared⁺&CHL⁺LB culture medium, transferring 2mL of the activated engineering bacteria according to 1% inoculum size into 200mL of the culture medium, culturing at 37 deg.C and 220rpm for 1h30min, 2h30min and 3h, adding 2mL of the bacteria into an EP tube, measuring ultraviolet absorption at 600nm wavelength with blank LB culture medium as controlAnd (6) harvesting.

After the positive engineering bacteria are activated and transferred and cultured, IPTG is added at a proper time point to induce the T7 promoter on the pET-28b-Cas9-His6 plasmid to start transcription. The research shows that OD₆₀₀Values of 0.6-0.8 are optimal induction start times. Therefore, the bacteria liquid at different time points after transfer is taken, the ultraviolet spectrophotometer detects the absorbance at 600nm to determine the optimal time, and the result is shown in Table 1, the OD at 2h30min₆₀₀The value was 0.769, which is the optimal IPTG induction start time.

TABLE 1 OD of the induced onset time bacterial liquid₆₀₀Results of value measurement (n ═ 3)

3. Screening for duration of inducible expression

Taking a sterilized empty test tube, picking the frozen pET-28b-Cas9 positive engineering bacteria, transferring to 5mL Kan⁺&CHL⁺The medium was incubated at 37 ℃ and 220rpm overnight for about 16 hours. A200 mL bottle of Kan was prepared⁺&CHL⁺And (3) LB liquid culture medium, transferring 2mL of bacterial liquid of the activated engineering bacteria according to 1% inoculum size into 200mL of culture medium, and culturing for 2h30min at 37 ℃ and 220 rpm. To each flask of the medium was added 10. mu.L of IPTG with a final concentration of 0.1mM, induced at 20 ℃ and maintained at 220rpm, and 2mL of the inoculum was removed every 2 hours from the addition of IPTG to an EP tube for 10 hours. In each of the 0 th, 2 th, 4 th, 6 th, 8 th and 10 th h samples, 10. mu.L of each sample was taken out to prepare SDS-PAGE samples, and the SDS-PAGE samples were analyzed.

The growth of the escherichia coli is divided into a delay phase, a logarithmic growth phase, a stationary phase and a decay phase, while the escherichia coli needs to grow continuously in the logarithmic growth phase, and escherichia coli cells need to be collected before the stationary phase, and then need to be screened. After the positive engineering bacteria are activated and transferred and cultured, SDS-PAGE analysis is carried out on the Cas9 protein expression amounts of 0, 2, 4, 6, 8 and 10h respectively after 2h and IPTG induction, the result is shown in figure 4, the Cas9 protein is not expressed at 0h, the protein expression amount at 2h is gradually increased until no growth occurs after 8h, therefore, 8h is taken as IPTG induction expression duration, and thalli are collected after 8 h.

4. Screening of bacteria-breaking conditions

Taking a sterilized empty test tube, picking the frozen pET-28b-Cas9 positive engineering bacteria, transferring to 5mL Kan⁺&CHL⁺The medium was incubated at 37 ℃ and 220rpm overnight for about 16 hours. A200 mL bottle of Kan was prepared⁺&CHL⁺And (3) LB liquid culture medium, transferring 2mL of bacterial liquid of the activated engineering bacteria according to 1% inoculum size into 200mL of culture medium, and culturing for 2h30min at 37 ℃ and 220 rpm. To each flask of the medium was added 10. mu.L of IPTG at a final concentration of 0.1mM, and induced at 220rpm at 20 ℃ for about 8 hours. The cells were collected by centrifugation at 8000g and 4 ℃ for 3min and resuspended in a suspension using a Binding Buffer. The conditions of the ultrasonic crusher are adjusted to be that the ultrasonic is started for 1s and stopped for 1s, the ultrasonic treatment is carried out for 15min, and the power is 700W, 600W and 500W respectively. And after the ultrasonic treatment is finished, 10 mu L of samples are taken out from the turbid total bacteria and the supernatant liquid of the turbid total bacteria respectively, and the optimum power of the Cas9 protein engineering bacteria for bacteria breaking is analyzed by SDS-PAGE.

Cas9 protein is stably expressed in the collected escherichia coli, and then the cell wall of the escherichia coli needs to be damaged to enable the content to flow out, so that the soluble Cas9 protein is dissolved in the supernatant. The power of ultrasonic bacteria breaking is the most important parameter for breaking the cell wall of the escherichia coli, protein carbonization can be caused when the power is too high, and the result of low protein yield can be caused when the cell wall of the escherichia coli is not completely broken when the power is too low. 700W, 600W and 500W are selected as the bacteria breaking power, and experimental phenomena are observed that protein carbonization is serious under the conditions of 700W and 600W, carbonized protein is basically not generated at 500W, and the bacteria breaking solution is relatively clear. This phenomenon is consistent with the results of SDS-PAGE analysis, and as shown in FIG. 5, the amount of protein in the supernatant was greatly reduced after disruption at 700W and 600W, while most of the protein was not disrupted at 500W and was dissolved in the supernatant, so 500W was used as the power for disruption.

5. Screening of the concentration of Heteroprotein imidazole eluate

Taking a sterilized empty test tube, picking the frozen pET-28b-Cas9 positive engineering bacteria, transferring to 5mL Kan⁺&CHL⁺The medium was incubated at 37 ℃ and 220rpm overnight for about 16 hours. A200 mL bottle of Kan was prepared⁺&CHL⁺Inoculating 2mL of the activated engineering bacteria according to 1% inoculum concentration in LB liquid culture mediumTo 200mL of the medium, the medium was incubated at 37 ℃ and 220rpm for 2h30 min. To each flask of the medium was added 10. mu.L of IPTG at a final concentration of 0.1mM, and induced at 220rpm at 20 ℃ for about 8 hours. The cells were collected by centrifugation at 8000g and 4 ℃ for 3min and resuspended in a suspension using a Binding Buffer. And adjusting the conditions of the ultrasonic crusher to start the ultrasonic for 1s and stop the ultrasonic for 1s, and carrying out ice bath ultrasonic for 15min under the power of 500W. Centrifuging at 10000g and 4 deg.C for 30min, collecting supernatant for purification, and temporarily storing at-20 deg.C.

Fixing Ni-NTA resin column on iron support, adding sterile water to clean resin, adding Binding Buffer balance resin column with 5 times column volume until it is stable, adding NiSO with 3 times column volume₄Blowing the resin column with the solution to make Ni²⁺The resin is fully bonded. Adding Binding Buffer cleaning resin until the resin is stable, calibrating the nucleic acid protein detector, and adding the sample to be purified. Placing the resin column at 4 deg.C to allow the target protein with His6 label to react with Ni in the resin²⁺After sufficient binding for 30min, the sample was slowly drained. Adding Binding Buffer to wash the resin column to make most of the hybrid protein fall off from the column, then washing the resin column according to the sequence of 30, 50, 70 and 90mM hybrid protein imidazole eluent, inoculating the effluent into an EP tube, taking 10 mu L of each effluent to prepare SDS-PAGE samples, and carrying out SDS-PAGE analysis on the optimal hybrid protein imidazole elution concentration.

The Cas9 protein sample to be purified contains a large amount of self-proteins, i.e., hybrid proteins, expressed internally by e. In the process of Ni-NTA resin column, after the sample is loaded, a part of protein flows out immediately when the sample is loaded, and a part of hybrid protein with weak Binding force flows out under the Binding Buffer elution, but the hybrid protein with stronger Binding force needs to be eluted by the low-concentration imidazole eluent. If the concentration of imidazole is too low, the purity of the finally obtained Cas9 protein cannot be eluted by the hybrid protein is low, and if the concentration of imidazole is too high, Cas9 protein bound in the resin column is eluted together to cause the loss of the target protein. As shown in FIG. 6, 30mM, 50 mM, 70 mM and 90mM of low concentration gradient imidazole are selected to elute the hybrid protein, and 30mM of imidazole eluent can elute most of the hybrid protein, while 50 mM, 70 mM and 90mM of concentration gradient imidazole carry away a small amount of hybrid protein but also carry away most of target protein, so that no target protein exists after 300mM of high concentration imidazole is eluted. Therefore, 300mM imidazole eluate was selected as the target protein eluate.

Purification with Ni chelate resin

Taking a sterilized empty test tube, picking the frozen pET-28b-Cas9 positive engineering bacteria, transferring to 5mL Kan⁺&CHL⁺The medium was incubated at 37 ℃ and 220rpm overnight for about 16 hours. A200 mL bottle of Kan was prepared⁺&CHL⁺And (3) LB liquid culture medium, transferring 2mL of bacterial liquid of the activated engineering bacteria according to 1% inoculum size into 200mL of culture medium, and culturing for 2h30min at 37 ℃ and 220 rpm. To each flask of the medium was added 10. mu.L of IPTG at a final concentration of 0.1mM, and induced at 220rpm at 20 ℃ for about 8 hours. The cells were collected by centrifugation at 8000g and 4 ℃ for 3min and resuspended in a suspension using a Binding Buffer. And adjusting the conditions of the ultrasonic crusher to start the ultrasonic for 1s and stop the ultrasonic for 1s, and carrying out ice bath ultrasonic for 15min under the power of 500W. Centrifuging at 10000g and 4 deg.C for 30min, collecting supernatant for purification, and temporarily storing at-20 deg.C.

Fixing Ni-NTA resin column on iron support, adding sterile water to clean resin, adding Binding Buffer balance resin column with 5 times column volume until it is stable, adding NiSO with 3 times column volume₄Blowing the resin column with the solution to make Ni²⁺The resin is fully bonded. Adding Binding Buffer cleaning resin until the resin is stable, calibrating the nucleic acid protein detector, and adding the sample to be purified. Placing the resin column at 4 deg.C to allow the target protein with His6 label to react with Ni in the resin²⁺After sufficient binding for 30min, the sample was slowly drained. Adding Binding Buffer to wash the resin column and 30mM imidazole eluent to elute the hybrid protein. Finally, a 300mM target protein imidazole eluent is added to elute the His 6-tagged Cas9 protein. Each effluent sample was taken 10. mu.L each to prepare an SDS-PAGE sample, which was analyzed by SDS-PAGE for the whole purification process.

Under the condition of screening various conditions in the expression and purification processes, the obtained purified sample finally passes through a Ni-NTA resin column, as shown in figure 7, most of protein flows out after penetrating through the resin column when the sample is loaded, a part of hybrid protein with weak Binding force flows out under Binding Buffer elution, hybrid protein with stronger Binding force flows out under 30mM imidazole eluent elution, and almost all Cas9 protein flows out together in 300mM imidazole eluent. The purity of the eluted Cas9 protein was higher.

7. Imidazole eluent ultrafiltration

Since the molecular weight of the Cas9 protein is about 160kDa, 300mM imidazole eluate was concentrated using a 50kDa ultrafiltration tube to remove a portion of free molecules such as heteroproteins and imidazole. Nano-100 was assayed for concentration at 280nm and 10. mu.L of the sample was prepared as SDS-PAGE, which analyzed the molecular weight and solubility of the final purified protein.

The Cas9 protein was highly pure in 300mM imidazole eluate with a small amount of low molecular weight hetero proteins. As shown in fig. 8, the Cas9 protein band obtained after ultrafiltration was about 160.0kDa, consistent with theoretical values, and was present in soluble form. As shown in Table 2, the purity obtained by Image J gray scanning calculation is 85.37%, the requirement of the experiment is met, and the product can be used as an important component in a CRISPR system for subsequent experiments.

Table 2 analysis of Cas9 protein purity by Image J

The amino acid and the corresponding nucleotide sequence of the Cas9 protein are shown as SEQ ID NO. 1.

8. The obtained Cas9 protein is subjected to circular dichroism spectrum, particle size, zeta potential and shear activity verification

(1) Circular dichroism spectrometry

The spectral properties of the Cas9 protein were determined using a circular dichroism spectrometer (Jasco J-180, Japan) in the extreme ultraviolet wavelength range 190nm to 260 nm. Cas9 protein samples were prepared at 0.05mg/mL, using a 1mm liquid bath, and accumulated 10 times.

The secondary structure of Cas9 protein was characterized by CD spectroscopy over the 190nm-260nm range, with the results shown in figure 9. The protein shows a positive peak near 195nm and negative peaks at 222nm and 208nm, which indicates that the Cas9 protein has partial alpha helical conformation, and shows that the Cas9 protein obtained by expression and purification is correctly folded and has a classical protein secondary structure.

(2) Measurement of particle diameter and zeta potential

A 1mg/mL Cas9 protein solution was prepared and filtered through a 0.45 μm syringe millipore filter. The protein particle size and zeta potential in the storage Buffer were determined by a malvern particle size potentiometer. Each sample was assayed in 3 replicates.

The particle size and potential of the Cas9 protein were examined by malvern particle size potentiostat, as shown in fig. 10, the DLS result for Cas9 protein was about 12.04nm, consistent with the theoretical value. As shown in table 3, Cas9 protein surface is positively charged, and Zeta potential is about +7.89 mV.

Table 3 particle size and Zeta potential determination of Cas9 protein (n ═ 3)

(3) Verification of cleavage activity of Cas9 protein

Cas9 protein and sgRNA are incubated for 10min at 37 ℃ at 100nM each, DNA purified product dsDNA 160ng is added into the complex, the total volume is 20 uL, the complex is kept still and digested for 2h at 37 ℃, and the agarose gel electrophoresis is carried out at 120V for 40min and 1% for verification.

Cas9 protein and sgRNA are combined to form a ribose protein complex, and dsDNA substrate shearing is carried out, as shown in figure 11, the Cas9 protein expressed by an escherichia coli expression system and purified by His6 tag nickel chelating resin has correct protein activity, namely gene editing performance.

Sequence listing

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Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

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His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

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Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

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His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

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Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

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Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

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Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

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Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1010 1015 1020

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser

1025 1030 1035 1040

Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu

1045 1050 1055

Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1060 1065 1070

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser

1075 1080 1085

Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly

1090 1095 1100

Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile

1105 1110 1115 1120

Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1125 1130 1135

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1140 1145 1150

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu LeuLeu Gly Ile Thr Ile

1155 1160 1165

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala

1170 1175 1180

Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys

1185 1190 1195 1200

Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1205 1210 1215

Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1220 1225 1230

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val

1265 1270 1275 1280

Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys

1285 1290 1295

His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1300 1305 1310

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp

1315 1320 1325

Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp

1330 1335 1340

Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile

1345 1350 1355 1360

Asp Leu Ser Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys

1365 1370 1375

Arg Lys Val Ala Ala Ala Leu Glu His His His His His His

1380 1385 1390

Claims

1. A method for expressing and purifying Cas9 protein, which is characterized in that: the method comprises the following steps:

step 1, transferring pET-28b-Cas9-His6 plasmid into host cellE.coliRosetta (DE3) pLysS, to obtain the recombinant strain Rosetta (DE3) pLysS;

2. The expression purification method according to claim 1, characterized in that: step 2 recombinant strains were transferred to 5 mLKan⁺&CHL⁺Culturing the culture medium at 37 ℃ and 220rpm for 16h for activation, measuring activated bacteria liquid according to 1 percent of inoculation amount after activation, and inoculating the activated bacteria liquid to Kan⁺&CHL⁺The medium was cultured at 37 ℃ and 220rpm for 2h30min, and then 0.1mM IPTG was added to induce expression.

3. The expression purification method according to claim 2, characterized in that: the temperature for inducing expression is 20 ℃ and the time is 8 h.

4. The expression purification method according to claim 1, characterized in that: and 3, ultrasonically breaking the bacteria under the conditions that the ultrasonic is started for 1s and stopped for 1s, and carrying out ice bath ultrasonic treatment for 15min under the power of 500W.

5. The expression purification method according to claim 1, characterized in that: the eluent in step 4 was 300mM imidazole.

6. The expression purification method according to claim 1, characterized in that: and 5, performing ultrafiltration concentration by adopting a 50kDa ultrafiltration tube.