A method for purifying a target protein from a solution

Abstract

The present application relates to the technical field of protein purification, and particularly relates to a method for purifying target protein from solution. The method for purifying target protein Cas9 from solution provided by the present application at least comprises the following steps: (a) adding polyethyleneimine into the solution, and collecting the precipitate; (b) washing with elution buffer, and collecting the supernatant; (c) ammonium sulfate gradient precipitation. The purification method of the present application can quickly prepare high-purity, correct-aggregate-form, good-DNA-cutting-activity, and tag-free Cas9 protein; moreover, the operation process of the purification method is simple, the repeatability is high, the method can be put into mass production, greatly reduces the purification cost of the tag-free Cas9 protein, and improves the purification efficiency.

Description

Technical Field

[0001] This invention relates to the field of protein purification technology, and more particularly to a method for purifying a target protein from solution. Background Technology

[0002] The CRISPR / Cas system is a natural immune system in prokaryotes, found in most bacteria and archaea. Currently, the most widely used CRISPR / Cas9 system in gene editing originates from *Streptococcus pyogenes* (…). Streptococcus pyogenes This gene editing system mainly consists of two parts: Cas9 protein and single-stranded guide RNA (sgRNA). Guided by the sgRNA, the Cas9 protein cleaves the target DNA, creating double-strand breaks. During subsequent intracellular repair, this alters the DNA sequence, achieving gene editing. Currently, Cas9 protein purification is mostly achieved by adding purification tags (such as His-tags). For example, Chinese patent application CN111893105A discloses a method for expressing and purifying Cas9 protein. This method involves selecting a Cas9 protein exogenous plasmid with a His6 tag, expressing it extensively in an *E. coli* expression system, and then using Ni... 2+ Cas9 protein is prepared by purification using metal chelating resins. Chinese patent application CN114410608A discloses a method for efficient expression and purification of Cas9 protein, which uses a 10×His label to enhance the affinity between the fusion protein and Ni-NTA resin, thereby facilitating the removal of contaminating nucleic acids through high-salt washing and improving purification efficiency. However, there are potential risks associated with the application of tagged Cas9 proteins in gene therapy or in vivo in animals.

[0003] With the continuous improvement and development of CRISPR / Cas9 technology, its application in gene therapy is increasing. To meet the requirements of more animal experiments and mitigate risks, Cas9 protein design has gradually shifted from early His-tag proteins to the development of tag-free proteins. Tag-free proteins, lacking tag amino acids, have a conformation closer to natural proteins and exhibit lower immunogenicity. As the application of CRISPR / Cas9 technology in in vivo gene editing increases, tag-free Cas9 proteins are gradually becoming the development trend in Cas9 protein preparation. Typically, the purification of tag-free proteins requires a combination of multiple types and steps of chromatography to obtain the target protein, such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, multimodal chromatography, and gel filtration chromatography, inevitably leading to a cumbersome purification process and low purification efficiency. Summary of the Invention

[0004] This invention provides a method for purifying the target protein Cas9 from solution.

[0005] This invention provides an efficient purification method for untagged Cas9 protein. By combining polyethyleneimine (PEI) precipitation with ammonium sulfate gradient precipitation, the purification efficiency of Cas9 protein is significantly improved. The untagged Cas9 protein obtained in this way has the correct aggregation form, a conformation closer to that of the natural protein, and good in vitro DNA cleavage activity.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] This invention provides a method for purifying the target protein Cas9 from solution, which includes at least the following steps:

[0008] (a) Add polyethyleneimine to the solution and collect the precipitate;

[0009] (b) Wash with elution buffer and collect the supernatant;

[0010] (c) Ammonium sulfate gradient precipitation.

[0011] Preferably, the method further includes at least one chromatography step, selected from: cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, or size exclusion chromatography.

[0012] In some embodiments of the present invention, the method for purifying the target protein Cas9 from solution includes one of the above-described chromatography techniques. Hydrophobic interaction chromatography is preferred, and requires only one step. This purification process is simple to operate and can rapidly prepare high-purity, correctly shaped aggregates of tag-free Cas9 protein with good DNA cleavage activity, greatly improving purification efficiency.

[0013] Preferably, the target protein Cas9 does not carry any purification tags.

[0014] In this invention, the purification tag includes all expression purification tags (peptides) known or unknown in the art, including but not limited to His tag, GST tag, MBP tag, CBD tag, Strep tag, Halo tag, SNAP tag, SUMO tag, NusA tag, TtxA tag, DsbA tag, etc.

[0015] In the above method, the polyethyleneimine is linear polyethyleneimine or branched polyethyleneimine.

[0016] Preferably, in step (a) above, polyethyleneimine is added to a working concentration of 0.6-0.8%.

[0017] Specifically, step (a) involves mixing the solution with polyethyleneimine to achieve a working concentration of 0.6-0.8% for the polyethyleneimine. After precipitation is complete, the mixture is centrifuged and the precipitate is collected.

[0018] In the above method, the solution is host cell lysis supernatant, host cell fermentation supernatant, or cell-free expression system.

[0019] Preferably, in step (b) above, the pH value of the elution buffer is 7-9, more preferably 7-8, and even more preferably 7.2-7.8.

[0020] Preferably, the elution buffer comprises: 600-1000 mM NaCl, 40-60 mM Tris-HCl, 15-25% glycerol, and 1-3 mM TCEP.

[0021] Preferably, the amount of the elution buffer is such that it is mixed with the precipitate collected in the previous step at a volume ratio of (1-2):1.

[0022] After washing with elution buffer, the precipitate was removed by centrifugation, and the supernatant was collected. This supernatant contained Cas9 protein with nucleic acid removed.

[0023] In step (c) above, the gradient precipitation involves first precipitating with ammonium sulfate at a final concentration of 26-28%, collecting the supernatant, and then precipitating with ammonium sulfate at a final concentration of 12-14%, and collecting the precipitate.

[0024] The first ammonium sulfate precipitation (with a final concentration of 26-28% ammonium sulfate) enables efficient precipitation of protein aggregates and their removal through separation. The second ammonium sulfate precipitation (with a final concentration of 12-14% ammonium sulfate) works well in conjunction with the first ammonium sulfate precipitation to precipitate the target protein Cas9, which has removed most of the protein aggregates. The high concentration of ammonium sulfate and residual PEI in the supernatant are then removed by centrifugation.

[0025] The final concentration of ammonium sulfate mentioned above refers to the final concentration of ammonium sulfate in the precipitation system.

[0026] Preferably, the precipitation time for each of the above-mentioned ammonium sulfate precipitation processes is 20-40 minutes.

[0027] In this invention, the supernatant or precipitate can be collected by centrifugation to separate the supernatant and precipitate. Preferably, centrifugation is performed at 9000-11000 g for 20-40 min.

[0028] Furthermore, the precipitate collected from the second ammonium sulfate precipitation was redissolved and then subjected to chromatography.

[0029] Preferably, the chromatography is hydrophobic interaction chromatography.

[0030] Specifically, the precipitate collected after ammonium sulfate gradient precipitation was reconstituted using buffer A, and then subjected to hydrophobic interaction chromatography, with elution using buffer B.

[0031] Buffer A consists of: 1-2M ammonium sulfate, 40-60mM Tris-HCl, and 1-3mM TCEP.

[0032] Buffer B consists of: 40-60 mM Tris-HCl, 1-3 mM TCEP.

[0033] Preferably, the pH of buffer A and buffer B is 7.2-7.8.

[0034] Preferably, the elution is linear elution.

[0035] More preferably, the linear elution volume is 10-20 cv, and the loading flow rate and elution flow rate are both 25-35 cm / h.

[0036] After the above elution, the peak components are collected to obtain the Cas9 protein.

[0037] In this invention, the host cell refers to a host cell capable of expressing the Cas9 protein, which contains a nucleic acid molecule encoding the Cas9 protein.

[0038] In this invention, the host cell is preferably *Escherichia coli*. Using the *E. coli* expression system, a large amount of soluble Cas9 recombinant protein can be fermented in a short time, achieving efficient soluble expression of Cas9 protein.

[0039] This invention does not have any particular restrictions on the specific strain of Escherichia coli, as long as it can express the Cas9 protein.

[0040] This invention does not impose any special restrictions on the amino acid sequence and source microorganism of the Cas9 protein, as long as it belongs to the Cas9 family of proteins.

[0041] In some embodiments of the present invention, the Uniprotocol number of the Cas9 protein is Q99ZW2-1. Cas9 proteins derived from other microorganisms, or Cas9 proteins or variants thereof having at least 50% similarity to the sequence shown in Q99ZW2-1, are also within the scope of protection of the present invention.

[0042] The present invention also provides the target protein Cas9 prepared by the method described above.

[0043] The beneficial effects of the present invention include at least the following: the method for purifying the target protein Cas9 from solution provided by the present invention can rapidly prepare high-purity, correctly aggregated, and DNA-cleaving-active untagged Cas9 protein; moreover, the purification method is simple to operate, highly reproducible, and can be put into mass production, which greatly reduces the purification cost of untagged Cas9 protein and improves the purification efficiency. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0045] Figure 1 This is a plasmid map of the expression vector pET28a-Cas9-Tag-free in Example 1 of the present invention.

[0046] Figure 2 This is an SDS-PAGE analysis diagram of the expression of tagless Cas9 protein by Escherichia coli fermentation in Example 2 of the present invention. The lanes from left to right are: M: protein marker, pre-induction: pre-induction whole protein, whole protein: post-induction whole protein, supernatant: lysed supernatant, and precipitate: lysed precipitate.

[0047] Figure 3 The image shows the SEC-MALS detection results of removing Cas9 protein aggregates using ammonium sulfate gradient precipitation in Example 3 of this invention. The peak time for Cas9 protein aggregates is around 11 min, and the peak time for monomers is around 15.3 min.

[0048] Figure 4 This is an SDS-PAGE analysis diagram of Cas9 protein purified by PEI precipitation in Example 3 of the present invention. The lanes from left to right are: M: protein marker, rupture supernatant, PEI precipitation supernatant: supernatant after PEI precipitation and centrifugation, elution supernatant: elution supernatant, and reconstitution supernatant: supernatant after reconstitution of Cas9 protein after ammonium sulfate precipitation.

[0049] Figure 5 This is a purified chromatogram of the target protein peak when hydrophobic chromatography was performed using AKTA prime in Example 4 of the present invention.

[0050] Figure 6 This is an SDS-PAGE image of the final Cas9 protein sample in Example 4 of the present invention, where M: protein marker.

[0051] Figure 7The image shows the SEC-MALS detection result of the final Cas9 protein sample in Example 4 of this invention. The results indicate that the peak elution time is around 15.3 min, the Cas9 protein is in monomeric form, and the monomeric content is greater than 95%.

[0052] Figure 8 This is an agarose gel electrophoresis image of the in vitro cleavage activity of Cas9 protein in Example 5 of the present invention. In the image, M represents the DNA marker, and the bands from top to bottom are 4500bp, 3000bp, 2000bp, 1200bp, 800bp, 500bp, and 200bp. 1 represents the control group with added dsDNA and sgRNA but no added Cas9, and 2 represents the experimental group with added Cas9, sgRNA, and dsDNA. The results show that the in vitro cleavage efficiency of unlabeled Cas9 protein is greater than 90%. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0054] Example 1 Construction of a tagless Cas9 protein recombinant expression vector

[0055] The construction method of the tag-free Cas9 protein recombinant expression vector (pET28a-Cas9-Tag-free) is as follows:

[0056] The nucleotide sequence encoding the tagless Cas9 protein (Uniprot accession number: Q99ZW2-1) was synthesized by Shanghai Bioengineering Co., Ltd., with an Nco I restriction endonuclease site added to the 5' end and a terminator and Nde I restriction endonuclease site added to the 3' end. The synthesized product was double-digested with Nco I and Nde I restriction endonucleases, followed by agarose gel electrophoresis, and the target fragment was recovered from the gel. The target fragment was ligated into pET28a(+) cells that had been double-digested with restriction endonucleases NcoⅠ and NdeⅠ to construct the recombinant pET28a-Cas9-Tag-free expression vector. This vector was transformed into *E. coli* DH5α and cultured at 37℃ for 16 h. Single colonies were then picked for plasmid extraction. The extracted plasmid was double-digested with restriction endonucleases BamHI and XhoⅠ and identified by agarose gel electrophoresis. Positive clones were sequenced, and clones with correct sequencing were selected for plasmid amplification and extraction. The extracted plasmids were aseptically filtered and stored at -20℃ for later use. The successfully constructed plasmid map is shown below.Figure 1 .

[0057] Example 2 Expression of tagless Cas9 recombinant protein in Escherichia coli

[0058] The pET28a-Cas9-Tag-free expression vector, stored at -20℃, was transformed into E. coli BL21(DE3) competent cells. After adding 1 mL of LB liquid medium, the cells were incubated at 37℃ in a shaker for 45 min. 200 μL of the culture was then spread onto Kansas cells. + Incubate resistant LB agar plates inverted at 37°C overnight. After overnight incubation, pick single colonies from the plate and transfer them to 20 mL of LB liquid medium, then add 50 μg / mL Kansin. + The culture was placed in a shaker at 37°C and incubated overnight. The bacterial culture that had grown to the logarithmic phase was then inoculated into 300 mL of TB medium in an Erlenmeyer flask with a baffle at the bottom and incubated at 37°C until OD500. 600 Once the concentration reaches 0.6–0.8, add IPTG to a final concentration of 0.2 mM, cool to 20°C, and incubate for 20 hours to terminate fermentation. Centrifuge the fermentation broth at 8000 g for 15 min and collect the precipitate.

[0059] The precipitate was resuspended in disruption buffer (purchased from ACROBIO), sonicated, and centrifuged at 10000 g for 30 min. The supernatant and precipitate after centrifugation were collected to prepare SDS-PAGE samples, which were then subjected to polyacrylamide gel electrophoresis (SDS-PAGE) together with the whole bacterial samples. The results are shown in the figure. Figure 2 .

[0060] Example 3: Isolation and purification of tagless Cas9 recombinant protein

[0061] The unlabeled Cas9 protein expressed in Escherichia coli in Example 2 was purified using the purification process developed in this invention. The specific method is as follows:

[0062] In Example 2, 5% (w / v) PEI solution was added to the supernatant after ultrasonic disruption and centrifugation while stirring to bring the working concentration of PEI in the solution to 0.8%. After complete precipitation, the mixture was centrifuged at 10000 g for 20 min, and the precipitate was collected. The precipitate was resuspended in equal volumes of elution buffer (900 mM NaCl, 50 mM Tris-HCl, 20% glycerol, 2 mM TCEP, pH 7.5) to elute nucleic acid and protein in the sample. The mixture was centrifuged at 10000 g at room temperature for 30 min, and the supernatant, which contained Cas9 protein contaminants removed, was collected. The supernatant collected after centrifugation was used to remove Cas9 protein aggregates and precipitate monomers using an ammonium sulfate gradient precipitation method. Ammonium sulfate solid was added to the supernatant while stirring until the final concentration was 27% (w / v) to precipitate protein aggregates. After precipitation for 30 min, the mixture was centrifuged at 10000 g at 4°C for 30 min. The precipitate was reconstituted and the protein aggregation morphology was determined using SEC-MALS. Figure 3 The results indicated that the precipitate consisted of protein aggregates, which were effectively removed by ammonium sulfate gradient precipitation. After centrifugation, the supernatant was collected, and the above operation was repeated with the addition of solid ammonium sulfate to a final concentration of 13% (w / v). After precipitation for 30 min, the mixture was centrifuged at 10000 g at 4°C for 30 min. The precipitate was collected, and it contained untagged Cas9 monomers after the removal of most of the protein aggregates.

[0063] The above processes involved SDS-PAGE analysis of the supernatant from bacterial cell disruption, supernatant after PEI precipitation, supernatant after elution with elution buffer, and supernatant from ammonium sulfate-precipitated protein reconstitution. The results are shown in [Figure number missing]. Figure 4 .

[0064] Furthermore, it has been verified that a PEI working concentration of 0.6% in the above method can achieve purification results comparable to those achieved with a PEI working concentration of 0.8%. Setting the concentrations of each component in the elution buffer within the following ranges—600-1000 mM NaCl, 40-60 mM Tris-HCl, 15-25% glycerol, 1-3 mM TCEP, and pH 7-9—all achieves purification results comparable to those obtained with the elution buffer used in this embodiment.

[0065] Example 4 Chromatographic purification of tag-free Cas9 recombinant protein

[0066] The unlabeled Cas9 monomer (precipitate collected after ammonium sulfate gradient precipitation) obtained using the purification method of Example 3 was subjected to hydrophobic chromatography (HIC) using Cytiva HiTrap Phenyl HP. The protein was reconstituted using buffer A (1.5M (NH4)2SO4, 50mM Tris-HCl, 2mM TCEP, pH 7.5). After reconstitution, the protein sample was filtered through a 0.2μm syringe filter to remove flocculent precipitate before hydrophobic chromatography. After equilibration with buffer A, the sample was loaded and linearly eluted using buffer B (50mM Tris-HCl, 2mM TCEP, pH 7.5) at a linear elution volume of 15 cv. The target protein monomer peak corresponded to a conductivity range of 110 mS / cm-91 mS / cm. Protein aggregates eluted after the monomer. The AKTA purification chromatogram of the eluted portion is shown in [Figure number missing]. Figure 5 .

[0067] The peak fractions collected by hydrophobic chromatography were concentrated by ultrafiltration, and after changing the buffer to the storage buffer, 50% glycerol was added to prepare the final protein sample. The sample was then subjected to SDS-PAGE electrophoresis, and the results are shown below. Figure 6 The monomer content of the final protein sample was determined by SEC-MALS analysis, and the results showed that the monomer content of the final purified Cas9 protein was greater than 95%. Figure 7 ).

[0068] The Cas9 protein purification method of the present invention is compared with the conventional chromatographic purification method for untagged Cas9 protein, as well as their protein yield and protein monomer ratio. As shown in Table 1, the PEI method for initial purification to capture untagged Cas9 protein not only has a high protein yield, but also greatly reduces the pressure of subsequent purification and impurity removal. Combined with ammonium sulfate gradient precipitation, protein aggregates can be removed easily and quickly. The overall purification method has significant advantages over conventional chromatographic purification, requiring fewer purification steps and achieving a higher protein yield.

[0069] Table 1

[0070]

[0071] Example 5: Validation of in vitro cleavage activity of tag-free Cas9 recombinant protein

[0072] The cleavage activity was detected using a commercial Cas9 in vitro digestion kit (Innovent Biologics Cas9 In Vitro Digestion Kit, catalog number PC1400). The experimental procedures were performed according to the product instructions. Agarose gel electrophoresis analysis of the results is shown below. Figure 8Lane 1 was the DNA marker, lane 2 was the control group with added dsDNA and sgRNA but no Cas9, and lane 3 was the experimental group with added Cas9, sgRNA and dsDNA. Electrophoresis results showed that the in vitro cleavage efficiency of untagged Cas9 protein was greater than 90%.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for purifying the target protein Cas9 from solution, characterized in that, It includes at least the following steps: (a) Add polyethyleneimine to the solution to a working concentration of 0.6-0.8% w / v and collect the precipitate; (b) Wash with elution buffer and collect the supernatant; (c) Ammonium sulfate gradient precipitation; The target protein Cas9 does not carry any purification tags; The Uniproto code number of the Cas9 is Q99ZW2-1; The elution buffer consists of: 600-1000 mM NaCl, 40-60 mM Tris-HCl, 15-25% glycerol, 1-3 mM TPCEP, and a pH of 7-9. The gradient precipitation process involves first precipitating with ammonium sulfate at a final concentration of 26-28% w / v, collecting the supernatant, and then precipitating with ammonium sulfate at a final concentration of 12-14% w / v, collecting the precipitate. The solution is a host cell lysis supernatant, a host cell fermentation supernatant, or a cell-free expression system.

2. The method according to claim 1, characterized in that, The method further includes at least one chromatography step, selected from cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, or size exclusion chromatography.

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