Preparation method and application of nuclease without protein tag

Abstract

The invention provides a preparation method and application of nuclease without a protein tag, and belongs to the field of protein engineering and biological pharmacy. The preparation method of the nuclease comprises the following steps that a sequence for coding the nuclease is connected to an expression vector, the expression vector is provided with elements such as a His protein tag, a TEV protease enzyme cutting site and a protein linker sequence, and the arrangement sequence of the elements is His tag-TEV enzyme cutting site-linker sequence-nuclease-linker sequence-TEV enzyme cutting site-His tag-His tag; expressing the fusion nuclease protein by using a prokaryotic expression system; and cutting the His tag on the fusion protein primarily purified by the Ni column by using TEV enzyme, and purifying and concentrating again to finally obtain the high-purity and high-enzyme-activity nuclease without the protein tag. The method is simple in step and low in production cost; and the nuclease yield, purity and enzyme activity are not inferior to those of other companies, so that the profit and competitiveness of the product can be increased by using the preparation process to produce the nuclease.

Description

Technical Field

[0001] The invention relates to a method for preparing a nuclease without a protein label and application thereof, and belongs to the field of protein engineering and biopharmaceuticals. Technical Background

[0002] Nuclease is a genetically engineered endonuclease derived from Serratia marcescens. Nuclease can degrade all forms of DNA and RNA (single-stranded, double-stranded, linear, circular) and has high specificity under a wide range of conditions. It can completely digest all nucleic acids into monophosphate oligonucleotides of 3-5 bases in length.

[0003] Nucleic acids have a wide range of uses. In the process of preparing biological products, the release of nucleic acids when cells or bacteria are lysed will produce viscosity. Nucleases can quickly hydrolyze nucleic acids, which can effectively reduce the viscosity of intermediate products, reduce processing time, and increase protein yield. For example, during chromatography purification, it can effectively avoid the reduction of the effective load of protein due to the adsorption of a large amount of nucleic acids on the chromatography medium, thereby reducing the purification yield. In experiments such as ELISA and immunoblotting analysis, it can improve resolution and recovery. In biological products, it can eliminate exogenous nucleic acid residues in recombinant proteins, vaccines and other biological products.

[0004] At present, although the preparation technology of nucleases has been relatively complete, and large biological companies have also had a relatively complete production system, large-scale high-precision instruments and equipment are required, and the production cost is high and the price is expensive. In order to reduce the preparation cost of nucleases, it is of great economic value to optimize the production process of nucleases. The Chinese patent "A method for preparing high-efficiency nucleases and its application" (CN 114854719 A) uses a yeast eukaryotic system to prepare high-efficiency nucleases. Although the system can achieve the expression of exogenous proteins, the proteins expressed in yeast are prone to form inclusion bodies, and the total amount of protein synthesized by yeast itself is more than that of Escherichia coli, which is not conducive to the subsequent purification steps; in addition, the growth rate of yeast is slower than that of Escherichia coli, thereby increasing the time cost. In summary, there is an urgent need for a method for preparing nucleases that is efficient and has low production cost, can best reduce protein structure and thus has the highest degradation efficiency. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for preparing a nuclease without a protein tag and its application. The specific steps of the preparation method of the nuclease are as follows: connecting the sequence encoding the nuclease to an expression vector, the expression vector carries elements such as a His protein tag, a TEV protease cleavage site and a protein linker (Linker) sequence, and the arrangement order of the elements is His tag-TEV enzyme cleavage site-Linker sequence-nuclease-Linker sequence-TEV enzyme cleavage site-His tag-His tag; the recombinant vector is transferred into the Escherichia coli DE3 strain, and then IPTG is used to induce the expression of exogenous proteins; the His tag on the fusion protein preliminarily purified by the Ni column is removed with TEV enzyme, and the protein is purified and concentrated again, and finally a protein tag-free nuclease with high purity and high enzyme activity is obtained. The method has simple steps, does not require advanced instruments, and the reagents used are cheap, so the production cost is low. The method for preparing nuclease provided by the present invention utilizes a natural signal peptide (SEQ ID NO: 2), so that the folded nuclease can be secreted outside the cell, and during the secretion process, the signal peptide will be automatically cut off, so as not to affect the structure of the nuclease. The method restores the structure of the nuclease to the maximum extent, does not contain protein tags and signal peptides, and the nuclease yield, purity and enzyme activity are no less than those of other companies. Therefore, the production of nucleases using this preparation process can increase the profit and competitiveness of the product.

[0006] In one aspect, the present invention provides a nuclease sequence, wherein the sequence comprises any one or more of a nuclease, a protein tag, a protease cleavage site and a protein linker sequence.

[0007] At present, the optimization of nuclease preparation methods is focused on the improvement of nuclease encoding sequences, the screening of signal peptides, the optimization of exogenous protein expression systems, etc., but no method has been proposed that can maximize the restoration of the structure of the nuclease itself and improve the purity of the protein. Therefore, the present invention provides and optimizes a nuclease sequence to prepare a nuclease without a protein tag. The introduction of a protease cleavage site can utilize the corresponding protease to remove the protein tag, and the linker sequence can improve the protease cleavage recovery rate.

[0008] Furthermore, the protein tag includes any one or more of His, MBP, GST, Myc, HA and SUMO; the sequence encoding the protease cleavage site includes one or more of LVPR▼GS (thrombin), DDDD▼K (enterokinase), LEVLFQ▼GP (PreScission protease), EXXYXQ▼(G / S) (TEV enzyme); the protein linker sequence includes one or more of (GGGGS)n, (EAAAK)n, (G)n and (XP)n, where n is an integer greater than or equal to 1.

[0009] It is understandable that the above-mentioned protein tags, protease cleavage sites and protein linker sequences can be arbitrarily matched according to the characteristics of the target protein to achieve the best preparation method.

[0010] In some specific embodiments, the protease cleavage site and protein linker sequence used are the TEV enzyme cleavage site (ENLYFQ▼G) and GGGGS, respectively;

[0011] Furthermore, the sequence element combination order is any one of protein tag-protease cleavage site-nuclease-protease cleavage site-protein tag, protein tag-protease cleavage site-protein linker sequence-nuclease-protein linker sequence-protease cleavage site-protein tag, or protein tag-protease cleavage site-protein linker sequence-nuclease-protein linker sequence-protease cleavage site-protein tag-protein tag.

[0012] Preferably, the sequence element combination order is protein tag-protease cleavage site-protein linker sequence-nuclease-protein linker sequence-protease cleavage site-protein tag-protein tag, which corresponds to the pET28b-NucA-Linker-TEV-His sequence in the present invention. The specific sequence of the sequence is shown in the sequence listing SEQ ID NO: 1.

[0013] In some specific embodiments, the introduction of TEV cleavage sites reduces the purification efficiency of nucleases. This may be because the introduction of TEV cleavage sites hinders the binding of nucleases to the nickel column, thereby reducing the purification efficiency. The addition of His tags increases the interaction between the nuclease and the nickel column, which can compensate for the effect of the TEV cleavage site on the purification efficiency.

[0014] In some preferred embodiments, a His tag is added to the C-terminus of the nuclease to prevent the newly added His tag from affecting the secretion of the nuclease; the number of added His tags should not exceed 1, that is, the nuclease that has not been cleaved by TEV has a maximum of 3 His tags, because the more His tags there are, the stronger the binding force between the nuclease and the Ni column, and the more difficult it is to elute, which in turn affects the yield of the protein. In summary, the best sequence element combination is His-TEV cleavage site-GGGGS-nuclease-GGGGS-TEV cleavage site-His-His.

[0015] On the other hand, the present invention provides a method for preparing a nuclease, which comprises preparing a nuclease using a sequence encoding the nuclease, wherein the sequence encoding the nuclease is as described above.

[0016] Furthermore, the steps of the preparation method are as follows:

[0017] A. constructing the sequence encoding the nuclease into an expression vector;

[0018] B. Induce protein expression and purify the protein;

[0019] C. Use protease to cleave nuclease protein;

[0020] D. Purify and concentrate the cleavage product in step C.

[0021] Furthermore, the protein expression in step B is a prokaryotic expression system or a eukaryotic expression system. In some specific embodiments, a prokaryotic expression system is selected.

[0022] Furthermore, the protein purification methods in step B and step C include any one or more of chromatography, precipitation, membrane separation technology and centrifugation.

[0023] Furthermore, the specific steps of the preparation method are as follows:

[0024] a. constructing the nucleotide sequence encoding the nuclease into the PET-28b prokaryotic expression vector;

[0025] b. The recombinant vector described in step a is transferred into an Escherichia coli expression strain, and after amplification and culture, the expression of the fusion nuclease protein is induced by IPTG, and purified by chromatography, wherein the Escherichia coli expression strain is any one or more of BL21 (DE3), Rosetta, and OrigamiB (DE3).

[0026] c. Use TEV protease to remove the His tag of the fusion nuclease protein purified in step b, and then use Ni column to adsorb the free His-TEV fusion protein in the enzyme cleavage reaction solution, so that the nuclease protein remains in the liquid;

[0027] d. Collect the liquid containing only nuclease in step c, and concentrate it by ultrafiltration.

[0028] On the other hand, the present invention provides a use of a nuclease sequence for preparing a reagent for improving the purity of a nuclease without a tag. The order of the combination of the nuclease sequence elements is protein tag-protease cleavage site-protein linker sequence-nuclease-protein linker sequence-protease cleavage site-protein tag-protein tag, and the nuclease sequence is named pET28b-NucA-Linker-TEV-His sequence in the present invention, and the specific sequence has the sequence shown in SEQ ID NO: 1 in the sequence table.

[0029] The beneficial effects of the present invention include:

[0030] 1. Through the use of some protein expression elements, such as His tags, flexible linkers, TEV restriction sites, etc., the structure of the nuclease can be temporarily and effectively changed to enable it to fold effectively, thereby obtaining a higher purity protein; then, the protein tag is removed to restore the structure of the nuclease itself to the maximum extent;

[0031] 2. The preparation method has a simple process, the required instruments are popular, and the reagents and consumables are cheap, so high-purity nuclease can be obtained, thereby reducing the production cost of nuclease and indirectly improving production profits and product competitiveness;

[0032] 3. The final nuclease product does not contain the His protein tag. The His tag is a commonly used protein tag. If the nuclease treatment object is subsequently used for pull-down, EMSA and other specific protein binding experiments, if the nuclease has a His tag, the experimental results may be unreliable;

[0033] 4. The preparation method provided by the present invention is also applicable to the production of other enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 : Partial schematic diagram of the expression vector designed in Example 1; His: 6 histidine tags; NucA: nuclease sequence; TEV: TEV proteolysis site; Linker: fusion protein linker sequence

[0036] Figure 2 : SDS-PAGE electrophoresis detection diagram of protein samples in Example 3; the sample in lane 1 is the concentrated replacement stock solution of the culture medium, and the sample in lane 2 is the flow-through sample, which refers to the liquid containing protein that flows out after the sample passes through the chromatography column and is not bound to the nickel column, and the lane 3 is the eluted protein sample; the protein sample in Figure A is expressed by the pET28b-NucA vector; the protein sample in Figure B is expressed by the pET28b-NucA-TEV vector; the protein sample in Figure C is expressed by the pET28b-NucA-Linker-TEV vector; the protein sample in Figure D is expressed by the pET28b-NucA-Linker-TEV-His vector; M: Marker

[0037] Figure 3: SDS-PAGE electrophoresis detection of nuclease after TEV protease cleavage, purification and concentration in Example 4; the protein sample in Figure A is expressed by the pET28b-NucA-TEV vector; the protein sample in Figure B is expressed by the pET28b-NucA-Linker-TEV vector; the protein sample in Figure C is expressed by the pET28b-NucA-Linker-TEV-His vector; M: Marker

[0038] Figure 4 : HPLC protein purity test of the tag-free nuclease prepared using the pET28b-NucA-Linker-TEV-His vector in Example 5

[0039] Figure 5 : Partial schematic diagram of the expression vector designed in Example 7; His: 6 histidine tags; NucA: nuclease sequence; TEV: TEV proteolysis site; Linker: fusion protein linker sequence; DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0041] Example 1: Design of expression vector

[0042] In this example, based on the universal expression vector pET28b, four vector structures containing nuclease encoding genes were designed by combining TEV restriction site (ENLYFQ▼G), His tag (HHHHHH) and fusion protein linker sequence (Linker) (GGGGS). Figure 1 ); the sequence was codon optimized and directly submitted to Shanghai Bioengineering Co., Ltd. for synthesis. The synthetic vector was pET28b+, and the connection sites were Nde I and Hind III. The final structure was as follows Figure 1 shown.

[0043] Example 2: Prokaryotic expression of foreign proteins

[0044] The four recombinant plasmids in Example 1 were respectively transformed into BL21 (DE3) strains; + After positive screening on the resistant LB plate, single clones were picked and cultured overnight on a shaking table. The next day, the bacterial solution was inoculated into a new Kan plate at a ratio of 1:100. + The culture was expanded in LB liquid medium with resistance to OD600 About 0.6; add IPTG to a final concentration of 1mM and induce overnight at 24°C; collect the culture medium, centrifuge at 4,500g for 30min; after centrifugation, the upper layer is the culture medium solution and the lower layer is the bacterial precipitate; because the N-terminus of the nuclease carries a signal peptide (SEQ ID NO:2, which will be removed later), is a secretory protein concentrated in the supernatant of the culture medium, so the supernatant was filtered twice using a microporous filtration membrane with a pore size of 0.6 μm (Tianjin Jinteng) and a microporous filtration membrane with a pore size of 0.45 μm (Tianjin Jinteng); since the size of the nuclease is about 30 kDa, a 10 kDa ultrafiltration membrane package (Millipore) was selected; before using the ultrafiltration membrane package, the membrane package was cleaned and disinfected with a 0.1 M sodium hydroxide solution, and then cleaned with ultrapure water, and then the solution to be treated (the supernatant containing the nuclease after filtration) was placed in an ultrafiltration device, and a certain pressure was applied to make the solution pass through the ultrafiltration membrane package, and the liquid at the reflux end, that is, the solution retaining the components greater than 10 kDa, was collected and concentrated to 100-200 mL, and an equilibrium buffer (20 mM Tris-HCl (pH = 8.0), 20 mM imidazole, 500 mM NaCl) to 400 mL, and then concentrated to 100-200 mL, repeated 3 times, and the final liquid at the reflux end was collected; finally, the membrane package was cleaned with ultrapure water and sodium hydroxide solution.

[0045] Example 3: Protein purification

[0046] In this example, the four fusion proteins in Example 2 were purified, and the specific steps were as follows: the AKTA equipment pipeline (equipment: AKTA Purifier 100) was rinsed with ultrapure water to make the conductivity and 280nm values ​​approach 0 and stable, and the column was balanced at room temperature; the connection flow rate was 3mL / min, and the 20% ethanol preservation solution in the column was rinsed with ultrapure water, and the volume of the ultrapure water for rinsing was about 3 times the volume of the column, until the baseline was stable; the column was balanced with 5-10 times the column volume of equilibrium buffer (20mM Tris-HCl (pH=8.0), 20mM imidazole, 500mM NaCl) until the conductivity remained stable; the sample was loaded (the sample was diluted with the equilibrium buffer in a ratio of 1:1), and the column was rinsed with the equilibrium buffer until the 280nm absorption peak value approached 0 and was stable; the elution buffer was increased in a gradient of 20mM Tris-HCl (pH=8.0), 500mM imidazole, 500mM NaCl) concentration (50%, 100%) to elute the target protein (nuclease), adjust the liquid flow rate in the column and the volume of the collected liquid according to the change of the 280nm peak, and collect the effluent under the corresponding elution peak, keep the volume of 100% elution buffer at 3 times the column volume, and then rinse with pure water until the conductivity and 280nm value tend to 0 and stabilize; rinse the column with about 3 times the column volume of 20% ethanol; and perform polyacrylamide gel electrophoresis on the collected eluate. For specific electrophoresis results, see Figure 2 .

[0047] Depend on Figure 2 From the AD graph, we can see that in lane 1, there is an obvious blue band in the range of 25kDa to 35kDa, indicating that four fusion proteins were successfully induced; compared with lane 1, the protein band in lane 3 is darker and larger, and the band is relatively single, that is, except for the target protein, there are basically no other protein bands, indicating that the target protein was successfully purified. The target band appeared in the second lane of Figure B and Figure C, while Figure 2 A and Figure 2 There is basically no nuclease in D, indicating that the expression of nuclease using vectors pET28b-NucA and pET28b-NucA-Linker-TEV-His (SEQ ID NO: 1) can improve the nickel column purification efficiency, while the nickel column purification efficiency of the two vectors pET28b-NucA-TEV and pET28b-NucA-Linker-TEV is lower. This may be because the introduction of the TEV restriction site hinders the binding of the nuclease to the nickel column, thereby reducing the purification efficiency. The addition of a His tag increases the interaction between the nuclease and the nickel column, which can then compensate for the effect of the TEV restriction site on the purification efficiency. In summary, the patents of the present invention can successfully induce and purify 4 target proteins, and the next step of the experiment can be carried out.

[0048] Example 4: TEV protease cleavage nuclease protein

[0049] The protein purified in Implementation 3 was dialyzed, and the dialysate (25mM Tris-HCl, 150mM NaCl, 0.1mM DTT (pH=8.0)) was replaced twice; 10mL 0.6mg / mL nuclease protein and 2.5mL 0.5mg / mL TEV protein were added to the dialysis bag, and the enzyme cleavage reaction was carried out in the dialysate at 27°C overnight; the enzyme cleavage reaction solution was taken out, and Binding buffer was added to dilute it at a volume ratio of 1:1, and then purified by Ni column, and the nuclease protein with His tag removed was in the flow-through liquid, and the TEV protein and His tag were bound to the Ni column; the nuclease protein in the concentrated flow-through liquid was replaced by a 10kDa Millipore membrane package into the storage solution (Tris-HCl, pH 7.4 10mM; NaCl 300mM; EDTA0.1mM; DTT 1mM; Glycerol (v / v) 50%) for storage, and 100% Elution Buffer was used to elute TEV protein and His tag to recover Ni column; 40 μL of concentrated nuclease protein was added to EP tube, and 10 μL of 5×SDS Loading Buffer was added, and boiled for 10 min. 30 μL was loaded for SDS-PAGE electrophoresis. Figure 3 .Depend on Figure 3 AC shows that nucleases without His tags can be obtained by cleaving three fusion proteins expressed from different vectors with TEV protease.

[0050] Example 5: Effects of different expression vectors on nuclease yield and purity

[0051] Although Example 4 demonstrates that the three vectors with TEV cleavage sites can all be used to prepare nucleases without His tags, the effects of different vector expressions on the yield and purity of nuclease proteins are still unknown, so this example explores this. The specific results are shown in Table 1, where Ni column purification efficiency = protease activity after Ni column purification / protease activity before Ni column purification * 100%, the protease activity method is described in Example 6, protein yield = protein content after TEV cleavage / Ni column purified protein amount * 100%, and protein purity is measured by HPLC.

[0052] Table 1 Effects of different expression vectors on nuclease yield and purity

[0053]

[0054] pET28b-NucA is the simplest expression vector. The obtained protein contains a His tag, which cannot be removed and the purity does not meet the requirements. A TEV protease cleavage site sequence is added between the His tag and the NucA protein sequence to obtain the pET28b-NucA-TEV expression vector. The fusion protein expressed by the vector can be hydrolyzed by the TEV protein to obtain a nuclease protein with higher purity without a tag. However, after the TEV protease cleavage site is added, the protein structure changes, the nickel column binding rate decreases (consistent with the results of Example 3), and the recovery rate decreases, and the TEV enzyme cleavage efficiency is average. A Linker sequence is added between TEV and the nuclease to obtain the pET28b-NucA-Linker-TEV expression vector. The Linker sequence makes the TEV protein The enzyme cleavage site and His tag can be better folded. Although the initial nickel column purification efficiency is similar to that without Linker, which is about 40%, the TEV enzyme cleavage recovery rate is increased to more than 80%. This is because the use of Linker can improve the folding and stability of the protein, so that the TEV protease cleavage site and the His tag can be correctly folded. The His tag is exposed at the end and is easy to bind to the nickel column; the pET28b-NucA-Linker-TEV-His expression vector introduces a His protein tag at the C-terminus of the nuclease on the basis of the pET28b-NucA-Linker-TEV vector. The initial nickel column purification efficiency of the fusion protein expressed by this vector is the highest, and the purification efficiency can reach more than 90%. The TEV enzyme cleavage recovery rate is more than 90%, and the purity can reach 99% ( Figure 4 ). In summary, it is most appropriate to use the pET28b-NucA-Linker-TEV-His (SEQ ID NO: 1) expression vector to express the nuclease sequence.

[0055] Example 6: Comparison of Nuclease Activity

[0056] Although Example 5 shows the effect of different vector expressions on the yield and purity of nuclease protein, the effect on nuclease activity is still unclear. This example compares the activity of the four nucleases prepared in the present invention and the nucleases purchased on the market. The general nuclease activity is defined as: under the reaction conditions of 37°C and pH=8.0, the amount of enzyme used to change the A260 absorption value of 2.625mL reaction system to 1.0 (equivalent to 37μg salmon sperm DNA being completely digested into oligonucleotides) within 30 minutes is defined as 1 activity unit (U). The specific experimental steps are as follows:

[0057] (1) Prepare reagent A: 50 mM Tris-HCl (pH = 8.0) containing 1 mM MgCl2 and 0.1 g / L BSA, volume 1 L;

[0058] (2) Prepare reagent B: Dissolve salmon sperm DNA in reagent A to obtain a salmon sperm DNA solution with a final concentration of 1 mg / mL. The volume should be adjusted according to the volume required for your sample.

[0059] (3) Nuclease sample dilution: Before diluting the nuclease sample, first use a micro-spectrophotometer (DeNovix DS-11+ ultra-micro UV spectrophotometer) to measure the initial protein concentration of each nuclease sample, and adjust the nuclease concentration to a consistent level (approximately 0.5-5 mg / mL) by adding sterile water; then dilute the nuclease sample according to the following steps:

[0060] Sample a: Take 1mL of reagent A and add 4μL of sample;

[0061] Sample b: Take 4 mL of reagent A and add 32 μL of sample a;

[0062] (4) Enzymatic reaction: 3 mL of salmon sperm DNA substrate was added to 150 μL of sample b and incubated at 37°C; 0.5 mL of the reaction solution with incubation time of 10 min, 20 min, 30 min, 50 min, and 80 min was taken into EP tubes respectively; 0.5 mL of 4% perchloric acid was added, mixed, and ice bathed for 30 to 60 min; 14,000 × g, 4°C centrifuged for 6 min, and the DNA content at A260 nm was measured by a micro-spectrophotometer (DeNovix DS-11+ ultra-micro-ultraviolet spectrophotometer). The amount of enzyme used to make the ΔA260 absorbance change 1.0 (equivalent to complete digestion of 37 ug salmon sperm DNA into oligonucleotides) in 2.625 ml reaction system within 30 minutes at 37°C and pH 8.0 was defined as one activity unit (U), and the nuclease activity was calculated. The specific results are shown in Table 2.

[0063] Table 2 Comparison of nuclease activity from different sources

[0064]

[0065] As can be seen from Table 2, the activities of all the nucleases of the present invention are not inferior to those of the nuclease products produced by other companies, indicating that the nuclease method provided by the present invention is feasible and has certain application value. At the same time, the preparation process is simple and fast, so the nuclease prepared by this method has certain market competitiveness; in addition, the nuclease prepared by the pET28b-NucA vector has the strongest activity, which may be because the protein purification is the simplest and fastest, and the protein is not easy to degrade, but the prepared nuclease has a His tag, which does not meet the purpose of the present invention-preparing nucleases without protein tags; although the nuclease activity expressed by the pET28b-NucA-TEV vector is slightly stronger than that of pET28b-NucA-Linker-TEV-His, the purification efficiency and protein purity of the former are not as good as the latter, so considering the above factors, it is most appropriate to use the pET28b-NucA-Linker-TEV-His vector to express nuclease.

[0066] Example 7: Effect of the position of the newly added His tag on the yield, purity and activity of nuclease

[0067] It can be seen from Examples 3 and 5 that adding a His tag can improve the purification efficiency of nucleases, but it is not clear whether the position of the newly added His tag will affect the purification efficiency. Therefore, in this example, a His tag is added to the N-terminus and / or the C-terminus of the nuclease ( Figure 5 ) to further optimize the purification efficiency of nuclease. The vector synthesis principle and process are the same as those described in Example 1, protein induction and purification are the same as those described in Examples 2 and 3, and the determination of protein content, purity and other indicators is the same as that described in Example 5. The specific results are shown in Table 3.

[0068] Table 3 Effects of different expression vectors on nuclease yield and purity

[0069]

[0070] As shown in Table 3, the position of the His tag does not affect the Ni column purification efficiency, protein yield and purity, but it will significantly affect the total amount of protein before purification, and then affect the total amount of protein obtained in the end, that is, the crude protein content is the highest when the new His tag is located at the C-terminus of the nuclease (pET28b-NucA-Linker-TEV-His), so the final nuclease content is also the highest. The reason why the position of the His tag affects the total amount of crude protein may be that there is a signal peptide at the N-terminus of the protein. The His tag added at the N-terminus affects the secretion of the protein, and then the total amount of nuclease in the supernatant decreases; it is worth noting that the addition of two His tags (pET28b-His-NucA-Linker-TEV-His) does not increase the Ni column purification efficiency. This may be because the interaction between the protein and the Ni column is too strong, resulting in the inability to completely elute the nuclease adsorbed on the Ni column with the elution buffer. In summary, the newly added His tag should be located at the C-terminus of the nuclease, and the number of newly added His tags should not exceed 1, that is, the number of His tags carried by the nuclease that has not been treated with TEV enzyme should not exceed 3; the pET28b-NucA-Linker-TEV-His (SEQ ID NO: 1) expression vector is most suitable for mass production of nucleases.

[0071] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A nuclease sequence, characterized in that The sequence includes any one or more of a nuclease, a protein tag, a protease cleavage site and a protein linker sequence.

2. The sequence according to claim 1, characterized in that The protein tag includes any one or more of His, MBP, GST, Myc, HA and SUMO; the sequence encoding the protease cleavage site includes one or more of LVPR▼GS, DDDD▼K, LEVLFQ▼GP, EXXYXQ▼G, EXXYXQ▼S; the protein linker sequence includes one or more of (GGGGS)n, (EAAAK)n, (G)n and (XP)n, where n is an integer greater than or equal to 1.

3. The sequence according to claim 2, characterized in that The sequence element combination order is any one of protein tag-protease cleavage site-nuclease-protease cleavage site-protein tag, protein tag-protease cleavage site-protein linker sequence-nuclease-protein linker sequence-protease cleavage site-protein tag, or protein tag-protease cleavage site-protein linker sequence-nuclease-protein linker sequence-protease cleavage site-protein tag-protein tag.

4. The sequence according to claim 3, characterized in that The sequence element combination order is protein tag-protease cleavage site-protein linker sequence-nuclease-protein linker sequence-protease cleavage site-protein tag-protein tag, and the specific sequence of the sequence is shown in the sequence table SEQ ID NO:

1.

5. A method for preparing a nuclease, characterized in that: The nuclease is prepared by using a sequence encoding the nuclease, wherein the sequence encoding the nuclease is as described in any one of claims 1 to 4.

6. The preparation method according to claim 5, characterized in that: The preparation method is as follows: A. constructing the sequence encoding the nuclease into an expression vector; B. Induce protein expression and purify the protein; C. Use protease to cleave nuclease protein; D. Purify and concentrate the cleavage product in step C.

7. The preparation method according to claim 6, characterized in that: The protein expression in step B is performed in a prokaryotic expression system or a eukaryotic expression system.

8. The preparation method according to claim 6, wherein the protein purification methods in step B and step C include any one or more of chromatography, precipitation, membrane separation technology and centrifugation.

9. The preparation method according to claim 6, wherein the specific steps of the preparation method are as follows: a. constructing the nucleotide sequence encoding the nuclease into the PET-28b prokaryotic expression vector; b. The recombinant vector described in step a is transferred into the E. coli expression strain, and after amplification, the expression of the fusion nuclease protein is induced with IPTG, and purified by chromatography. c. Use TEV protease to remove the His tag of the fusion nuclease protein purified in step b, and then use Ni column to adsorb the free His-TEV fusion protein in the enzyme cleavage reaction solution, so that the nuclease protein remains in the liquid; d. Collect the liquid containing only nuclease in step c, and concentrate it by ultrafiltration.

10. A use of a nuclease sequence for preparing a reagent for improving the purity of an untagged nuclease, characterized in that: The combination order of the nuclease sequence elements is protein tag-protease cleavage site-protein linker sequence-nuclease-protein linker sequence-protease cleavage site-protein tag-protein tag, and the specific sequence of the nuclease sequence has the sequence shown in sequence table SEQ ID NO: 1.

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