CN117801056A - Method for purifying target protein from solution - Google Patents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
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- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to the technical field of protein purification, in particular to a method for purifying target protein from solution. The method for purifying the target protein Cas9 from the solution at least comprises the following steps: (a) adding polyethylenimine to the solution and collecting the precipitate; (b) washing with elution buffer and collecting the supernatant; (c) ammonium sulfate gradient precipitation; the target protein Cas9 does not carry any purification tag. The purification method can rapidly prepare the label-free Cas9 protein with high purity, correct aggregate form and good DNA cleavage activity; the purification method has the advantages of simple operation process and high repeatability, can be put into mass production, greatly reduces the purification cost of the non-tag Cas9 protein, and improves the purification efficiency.
Description
Technical Field
The invention is a divisional application of patent application with application number 202310780220X and application number 2023, 6, 29 and the name of "a method for purifying target protein from solution". The invention relates to the technical field of protein purification, in particular to a method for purifying target protein from solution.
Background
The CRISPR/Cas system is a natural immune system of prokaryotes, present in most bacteria and archaea. Currently, the most widely used CRISPR/Cas9 system in gene editing is derived from streptococcus pyogenes (Streptococcus pyogenes). The gene editing system mainly comprises a Cas9 protein and a single guide RNA (sgRNA), wherein the Cas9 protein cuts target site DNA under the guiding action of the sgRNA and generates double strand break, and the change of DNA sequence is realized in the subsequent intracellular repair process, so that the gene editing effect is achieved. Currently purification of Cas9 proteins is mostly done by way of adding purification tags (e.g. His-tag, etc.), such as: chinese patent application CN111893105A discloses a method for expressing and purifying Cas9 protein, which comprises selecting Cas9 protein exogenous plasmid with His6 tag to express in large quantity in E.coli expression system and adopting Ni 2+ And purifying the metal chelate resin to prepare the Cas9 protein. Chinese patent application CN114410608A discloses a method for efficiently expressing and purifying Cas9 protein, by using 10×his tag to increase affinity of fusion protein to Ni-NTA resin, to facilitate high salt washing to remove contaminated nucleic acid, and to increase purification effect. However, tagged Cas9 proteins present a potential risk in the field of gene therapy or in vivo animal applications.
With the continuous perfect development of CRISPR/Cas9 technology, the CRISPR/Cas9 technology has increasingly been applied to the field of gene therapy. In order to meet the requirements of more animal experiments and avoid risks, the design of Cas9 proteins is also gradually shifted from early His-Tag tagged proteins to Tag-free untagged protein development. Unlabeled proteins are more conformational to natural proteins and less immunogenic due to the absence of labeled amino acids. As the application of CRISPR/Cas9 technology in vivo gene editing increases, unlabeled Cas9 protein also gradually becomes a development trend of Cas9 protein preparation. Generally, purification of a tag-free protein requires a combination of various types and steps of chromatography to obtain a target protein, such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, multi-mode chromatography, gel filtration chromatography, and the like, which inevitably results in complicated purification processes and low purification efficiency.
Disclosure of Invention
The present invention provides a method for purifying a target protein Cas9 from a solution.
The invention provides a high-efficiency purification method for the non-tag Cas9 protein, which is characterized in that polyethylene imine (PEI) precipitation and ammonium sulfate gradient precipitation are combined, so that the purification efficiency of the Cas9 protein is remarkably improved, and the prepared non-tag Cas9 protein has a correct aggregation form, a conformation which is closer to that of a natural protein and good DNA in-vitro cutting activity.
Specifically, the invention provides the following technical scheme:
the invention provides a method for purifying a target protein Cas9 from a solution, which at least comprises the following steps:
(a) Adding polyethyleneimine into the solution, and collecting the precipitate;
(b) Washing with elution buffer, and collecting supernatant;
(c) Ammonium sulfate gradient precipitation.
Preferably, the method further comprises at least one step of chromatography selected from: cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography or size exclusion chromatography.
In some embodiments of the invention, the method of purifying the target protein Cas9 from solution comprises one of the chromatographic techniques described above. Hydrophobic interaction chromatography is preferred, and only one-step chromatography treatment is needed, so that the purification process is simple to operate, the unlabeled Cas9 protein with high purity, correct aggregate form and good DNA cleavage activity can be rapidly prepared, and the purification efficiency is greatly improved.
Preferably, the target protein Cas9 does not carry any purification tag.
In the present invention, the purification tag includes all expression purification tags (polypeptides) known or unknown in the art, including but not limited to His tags, GST tags, MBP tags, CBD tags, strep tags, halo tags, SNAP tags, SUMO tags, nusA tags, ttxA tags, dsbA tags, and the like.
In the above method, the polyethylenimine is linear polyethylenimine or branched polyethylenimine.
Preferably, in the step (a), polyethyleneimine is added to a working concentration of 0.6 to 0.8%.
Specifically, step (a) is to mix the solution with polyethyleneimine so that the polyethyleneimine reaches a working concentration of 0.6-0.8%, and after precipitation is completed, centrifuging and collecting the precipitate.
In the above method, the solution is a host cell disruption supernatant, a host cell fermentation supernatant, or a cell-free expression system.
Preferably, in step (b) above, the pH of the elution buffer is 7-9, preferably 7-8, more preferably 7.2-7.8.
Preferably, the elution buffer comprises: 600-1000mM NaCl,40-60mM Tris-HCl,15-25% glycerol, 1-3mM TCEP.
Preferably, the elution buffer is used in an amount such that the volume ratio to the precipitate collected in the previous step is (1-2): 1.
After washing with elution buffer, the precipitate was removed by centrifugation and the supernatant was collected. The supernatant contained the Cas9 protein with the nucleic acid removed.
In the step (c), the gradient precipitation is that ammonium sulfate with the final concentration of 26-28% is adopted for precipitation, then supernatant is collected, and ammonium sulfate with the final concentration of 12-14% is adopted for precipitation, and then precipitation is collected.
The first ammonium sulfate precipitation (the precipitation of ammonium sulfate with the final concentration of 26-28%) can enable the protein aggregate to be efficiently precipitated and removed through separation, the second ammonium sulfate precipitation (the precipitation of ammonium sulfate with the final concentration of 12-14%) can well cooperate with the first ammonium sulfate precipitation, the target protein Cas9 from which most of the protein aggregate is removed is precipitated, and the high concentration of ammonium sulfate and residual PEI in the supernatant are removed through centrifugation.
The final concentration of ammonium sulfate described above is the final concentration of ammonium sulfate in the precipitation system.
Preferably, the time for each ammonium sulfate precipitation is 20-40min.
In the present invention, the supernatant or precipitate may be collected by centrifugation. Preferably centrifuged at 9000-11000g for 20-40min.
Further, the precipitate collected by the second ammonium sulfate precipitation is redissolved and then subjected to chromatography.
Preferably, the chromatography is hydrophobic interaction chromatography.
Specifically, adopting buffer A to carry out redissolution on the collected precipitate after ammonium sulfate gradient precipitation, then carrying out hydrophobic interaction chromatography, and adopting buffer B to carry out elution;
wherein buffer a comprises: 1-2M ammonium sulfate, 40-60mM Tris-HCl,1-3mM TCEP.
Buffer B included: 40-60mM Tris-HCl,1-3mM TCEP.
Preferably, the pH of buffer A and buffer B is 7.2-7.8.
Preferably, the elution is a linear elution.
Further preferably, the volume of the linear elution is 10-20cv, and the loading flow rate and the elution flow rate are 25-35cm/h.
And collecting peak components after the elution, thus obtaining the Cas9 protein.
In the present invention, the host cell refers to a host cell capable of expressing Cas9 protein, which contains a nucleic acid molecule encoding Cas9 protein.
In the present invention, the host cell is preferably E.coli. A large amount of soluble Cas9 recombinant proteins can be prepared by fermenting an escherichia coli expression system in a short time, so that high-efficiency soluble expression of the Cas9 proteins is realized.
The specific strain of E.coli is not particularly limited as long as it can express the Cas9 protein.
The amino acid sequence of the Cas9 protein and the source microorganism are not particularly limited as long as the Cas9 family protein is included in the present invention.
In some embodiments of the invention, the Cas9 protein has Uniprot accession number Q99ZW2-1. Cas9 proteins derived from other microorganisms or Cas9 proteins having at least 50% similarity to the sequence shown in Q99ZW2-1 or variants thereof are also within the scope of the invention.
The invention also provides the target protein Cas9 prepared by the method.
The beneficial effects of the invention at least comprise: the method for purifying the target protein Cas9 from the solution can rapidly prepare the label-free Cas9 protein with high purity, correct aggregate form and good DNA cutting activity; the purification method has the advantages of simple operation process and high repeatability, can be put into mass production, greatly reduces the purification cost of the non-tag Cas9 protein, and improves the purification efficiency.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a plasmid map of expression vector pET28a-Cas9-Tag-free in example 1 of the present invention.
FIG. 2 is a SDS-PAGE analysis of E.coli fermentatively expressing unlabeled Cas9 protein according to example 2 of the present invention, wherein the lanes are, in order from left to right: m: protein Marker, before induction: pre-induction holoprotein, holoprotein: whole protein, supernatant after induction: bacterial breaking supernatant and precipitation: and (5) bacterial breaking and sedimentation.
Fig. 3 is a graph of SEC-MALS detection results using ammonium sulfate gradient precipitation to remove Cas9 protein aggregates in example 3 of the present invention, wherein Cas9 protein aggregates have a peak time of about 11min and monomers have a peak time of about 15.3 min.
FIG. 4 is a SDS-PAGE analysis of PEI precipitation purified Cas9 protein of example 3 of the present invention, wherein the lanes are, in order from left to right: m: protein Marker, broken: bacterial supernatant, PEI pellet: supernatant after PEI precipitation and centrifugation, on elution: eluting supernatant, redissolving: cas9 protein redissolved supernatant after ammonium sulfate precipitation.
FIG. 5 is a purification chromatogram of the peak portion of the target protein when the AKTAprime is used for hydrophobic chromatography in example 4 of the present invention.
FIG. 6 is a SDS-PAGE diagram of final sample of Cas9 protein in example 4 of the present invention, wherein M: protein Marker.
Fig. 7 is a SEC-MALS detection diagram of a final sample of Cas9 protein in example 4 of the present invention, and the result shows that the peak time is about 15.3min, and Cas9 protein is in a monomer form, and the monomer ratio is greater than 95%.
FIG. 8 is a graph of agarose gel electrophoresis detection of in vitro cleavage activity of Cas9 protein in example 5 of the present invention, wherein M: DNAMmarker, bands from top to bottom are 4500bp, 3000bp, 2000bp, 1200bp, 800bp, 500bp, 200bp,1: control group with dsDNA and sgRNA added, but no Cas9 added, 2: experimental groups added with Cas9, sgRNA and dsDNA show that the in vitro cleavage efficiency of the unlabeled Cas9 protein is more than 90%.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 construction of untagged Cas9 protein recombinant expression vector
The construction method of the unlabeled Cas9 protein recombinant expression vector (pET 28a-Cas 9-Tag-free) is as follows:
a nucleotide sequence corresponding to an amino acid sequence of a coded non-tagged Cas9 protein (Uniprot accession number: Q99ZW 2-1) is synthesized by Shanghai bioengineering limited company, and a restriction enzyme NcoI cleavage site is added at the 5 'end, a terminator and a restriction enzyme NdeI cleavage site are added at the 3' end. The synthesized product is digested with restriction enzymes NcoI and NdeI, and then subjected to agarose gel electrophoresis, and the desired fragment is recovered by cutting. The target fragment is respectively connected with pET28a (+) which is also subjected to double enzyme digestion by restriction enzymes NcoI and NdeI, a recombinant pET28a-Cas9-Tag-free expression vector is constructed, E.coli DH5 alpha is transformed, single colony is selected for plasmid extraction after 16h culture at 37 ℃, agarose gel electrophoresis identification is carried out on the extracted plasmid after double enzyme digestion by restriction enzymes BamHI and XhoI, positive clone sequencing is carried out, clone with correct sequencing is selected for amplified extraction of the plasmid, the extracted plasmid is subjected to aseptic filtration, and the plasmid is preserved at-20 ℃ for standby, and the constructed plasmid map is shown in figure 1.
EXAMPLE 2 expression of unlabeled Cas9 recombinant proteins in E.coli
Transferring the pET28a-Cas9-Tag-free expression vector stored at the temperature of minus 20 ℃ into E.coli BL21 (DE 3) competent cells, adding 1mL of LB liquid medium, placing the mixture in a shaking table at the temperature of 37 ℃ for 45min, and coating 200 mu L of the mixture on Kan + On a resistant LB solid plate, the plate was inverted and incubated overnight in an incubator at 37 ℃. After overnight incubation, the plate was picked up from a monoclonal colony to 20mL of LB liquid medium, and 50. Mu.g/mL Kan final concentration was added + Culturing overnight at 37deg.C, inoculating bacterial liquid growing to logarithmic phase into 300mL TB culture medium, selecting triangular conical flask with baffle at bottom, culturing at 37deg.C to OD 600 Reaching 0.6-0.8, adding IPTG with the final concentration of 0.2mM, cooling to 20 ℃ and culturing for 20 hours, and ending fermentation. The fermentation broth was centrifuged at 8000g for 15min and the pellet was collected after centrifugation.
The pellet was resuspended in a proportion of disruption buffer (available from Acronbio), sonicated, centrifuged at 10000g for 30min, and the supernatant after the cell disruption centrifugation was collected and the pellet was subjected to SDS-PAGE to prepare a SDS-PAGE sample, which was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) along with the whole cell sample, as shown in FIG. 2.
EXAMPLE 3 isolation and purification of untagged Cas9 recombinant proteins
The purification process developed by the invention is used for purifying the unlabeled Cas9 protein expressed by escherichia coli in the embodiment 2, and the specific method is as follows:
5% (w/v) PEI solution was added to the centrifuged supernatant of example 2 with stirring to give a working concentration of 0.8% PEI in the solution, and after completion of precipitation, the mixture was centrifuged at 10000g for 20min, and the precipitate was collected. The nucleic acid precipitate and protein were eluted from the sample by isovolumetric resuspension of the precipitate using elution buffer (900mM NaCl,50mM Tris-HCl,20% glycerol, 2mm tcep, ph 7.5), centrifuged at 10000g for 30min at room temperature, and the supernatant was collected, which contained Cas9 protein with contamination removed from nucleic acid, etc. Removing Cas9 protein aggregates and precipitating monomers from supernatant collected after centrifugation by adopting an ammonium sulfate gradient precipitation method, and adding ammonium sulfate solid to the supernatant while stirring to a final concentration of 27% (w/v) to precipitate protein aggregates; after 30min of precipitation, the precipitate was centrifuged at 10000g at 4℃for 30min, and the protein aggregate morphology was determined by SEC-MALS after reconstitution of the precipitate, and FIG. 3 shows that the precipitate component was protein aggregate, which was effectively removed by ammonium sulfate gradient precipitation. After centrifugation the supernatant was collected, solid ammonium sulphate was added to a final concentration of 13% (w/v) and after 30min of precipitation, centrifuged at 10000g for 30min at 4℃and the precipitate was collected, which contained unlabeled Cas9 monomer after removal of most protein aggregates.
In the above procedure, SDS-PAGE was performed on samples of the cell disruption supernatant, PEI-precipitated supernatant, elution buffer-eluted supernatant, and ammonium sulfate precipitated protein redissolved supernatant, respectively, and the results are shown in FIG. 4.
In addition, it was confirmed that the purification effect equivalent to that obtained when the PEI working concentration was 0.8% was obtained even when the PEI working concentration was 0.6% in the above-mentioned method. The concentration of each component in the elution buffer was set within the following range: the purification effect equivalent to the elution buffer used in this example was obtained with 600-1000mM NaCl,40-60mM Tris-HCl,15-25% glycerol, 1-3mM TCEP, pH 7-9.
EXAMPLE 4 chromatographic purification of untagged Cas9 recombinant proteins
The unlabeled Cas9 monomer (precipitate collected after ammonium sulfate gradient precipitation) prepared by the purification method of example 3 was subjected to hydrophobic chromatography (HIC) using Cytiva HiTrap Phenyl HP using buffer a (1.5M (NH) 4 ) 2 SO 4 50mM Tris-HCl,2mM TCEP,pH 7.5), filtering the re-dissolved protein sample with a 0.2 μm needle filter to remove flocculent precipitate, performing hydrophobic chromatography, and balancing with buffer AThe sample was then loaded and eluted with a linear elution volume of 15cv using buffer B (50 mM Tris-HCl,2mM TCEP,pH 7.5). The peak of the target protein monomer corresponds to the electric conduction range of 110mS/cm-91mS/cm, the protein aggregate is subjected to peak generation after the monomer, and the AKTA purification chromatogram of the peak generation part is shown in figure 5.
The peak fraction collected by hydrophobic chromatography was concentrated by ultrafiltration, and after changing the solution to a preservation buffer, glycerol was added at a final concentration of 50% to prepare a final protein sample, which was sampled and subjected to SDS-PAGE, the results of which are shown in FIG. 6. The protein end samples were subjected to SEC-MALS detection monomer duty cycle, which indicated that the final Cas9 protein purification monomer duty cycle was greater than 95% (fig. 7).
Comparing the Cas9 protein purification method of the present invention with the conventional chromatographic purification method of the unlabeled Cas9 protein, the protein yield and the protein monomer ratio, as shown in table 1, the use of PEI for preliminary purification to capture the unlabeled Cas9 protein not only has higher protein yield, but also can greatly relieve the pressure of subsequent purification and impurity removal, and the combination of ammonium sulfate gradient precipitation can simply, conveniently and rapidly remove protein aggregates, and compared with the conventional chromatographic purification, the whole purification method adopts fewer purification steps, can realize higher protein yield, and has obvious advantages.
TABLE 1
Example 5 in vitro cleavage Activity validation of unlabeled Cas9 recombinant proteins
The cleavage activity assay was performed using a commercial Cas9 in vitro cleavage kit (ex. Cas9 in vitro cleavage kit, cat# PC1400 from the intel flourishing industry), the experimental procedure was performed according to the product instructions and the agarose gel electrophoresis analysis of the assay results is shown in fig. 8. The 1 st lane is a DNA Marker, the 2 nd lane is a control group added with dsDNA and sgRNA but not added with Cas9, the 3 rd lane is an experimental group added with Cas9, sgRNA and dsDNA, and the electrophoresis result shows that the in vitro cleavage efficiency of the unlabeled Cas9 protein is more than 90%.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (7)
1. A method for purifying a protein of interest Cas9 from a solution, characterized in that it comprises at least the following steps:
(a) Adding polyethyleneimine into the solution, and collecting the precipitate;
(b) Washing with elution buffer, and collecting supernatant;
(c) Ammonium sulfate gradient precipitation;
the target protein Cas9 does not carry any purification tag.
2. The method of claim 1, further comprising at least one chromatography step selected from the group consisting of cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography.
3. The process according to claim 1 or 2, wherein in step (a) polyethylenimine is added to a working concentration of 0.6-0.8%.
4. The method of claim 1 or 2, wherein the solution is a host cell disruption supernatant, a host cell fermentation supernatant, or a cell-free expression system.
5. The method according to claim 1 or 2, wherein the pH of the elution buffer is 7-9.
6. The method according to claim 1 or 2, wherein in step (c), the gradient precipitation is performed by first precipitating with ammonium sulfate having a final concentration of 26-28%, then collecting the supernatant, and then precipitating with ammonium sulfate having a final concentration of 12-14%, and collecting the precipitate.
7. A Cas9 protein prepared by the method of any one of claims 1-6.
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