Application of DNA binding protein Mse7 in removal of host nucleic acid in recombinant protein

Abstract

The invention discloses application of DNA (deoxyribonucleic acid) binding protein Mse7 in removal of host nucleic acid in recombinant protein, a chromatography medium based on the DNA binding protein Mse7 comprises biotin ligase, streptavidin agarose gel and the DNA binding protein Mse7, and the DNA binding protein Mse7 is connected with an Avi-tag label. The method comprises the following steps: co-expressing biotin ligase and DNA (Deoxyribose Nucleic Acid) binding protein in vivo to obtain biotinylated Mse7 protein; purifying and concentrating; and finally, incubating and combining the Mse7 with streptavidin agarose gel to form a new chromatographic filler. The DNA binding protein Mse7 serves as a novel chromatography medium to be applied to removal of host nucleic acid in recombinant protein, host DNA can be effectively removed, the purity of a product is improved, and compared with a traditional method, the technology has higher purification efficiency and recovery rate.

Description

Technical Field

[0001] The invention belongs to the biological field, and in particular relates to the application of DNA binding protein Mse7 in removing host nucleic acid in recombinant protein. Background Art

[0002] In the production of recombinant proteins, the problem of host nucleic acid residues has always been an important challenge. These residual nucleic acid fragments may bring potential safety risks, including the risk of tumorigenicity, infectivity, immunogenicity and mutagenicity. Therefore, strict control of host nucleic acid residues in tool enzymes is a top priority for the safety and efficacy of biological products.

[0003] There are two main methods for removing host nucleic acids from recombinant proteins in the market: ion exchange chromatography and enzymatic method. Ion exchange chromatography uses the characteristics of DNA with a large amount of negative charge to remove it by adsorption by anion exchangers. However, this anion filler purification process takes a certain amount of time, which may affect the preparation efficiency of biological tool enzymes; secondly, the anion filler purification process requires strict control of operating conditions, such as pH, temperature, time, etc., to avoid affecting the purification effect. The enzyme method mainly relies on human deoxyribonuclease I and universal nuclease, which can efficiently remove nucleic acid residues. Although it can degrade DNA, it is not specific to host DNA. This means that while degrading host DNA, it may also degrade the DNA of the enzyme itself or other related molecules, which may affect the activity and function of the enzyme. Moreover, improper use of the enzymatic method may pose potential safety risks to operators and the environment. Therefore, when using universal nucleases for purification, it is necessary to weigh the pros and cons and take appropriate measures to overcome these shortcomings.

[0004] Traditional methods have the problems of cumbersome operation, long time consumption, low efficiency, affecting the activity function of biological enzymes and potential safety hazards to people. Therefore, it is urgent to explore a method that is comparable to anion host removal, short time consumption, high efficiency, simple and safe operation, does not affect the product quality of the enzyme, and can effectively remove host DNA residues in biological tool enzymes. This new method is expected to improve the safety and effectiveness of biological tool enzymes and promote the development of biotechnology. Summary of the invention

[0005] Purpose of the invention: In order to solve the above-mentioned technical problems, the present invention provides a chromatography medium based on the DNA binding protein Mse7, which does not adsorb RI protein, but only binds to the host nucleic acid in the RI protein solution, effectively removing the host DNA residues in the biological tool enzyme, with short time consumption, high efficiency, simple and safe operation, and improving the safety and effectiveness of the biological tool enzyme.

[0006] The present invention also provides the use of a chromatography medium based on the DNA binding protein Mse7 in removing host nucleic acid from recombinant proteins.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a chromatography medium based on DNA binding protein Mse7, wherein the chromatography medium comprises biotin ligase, streptavidin agarose gel and DNA binding protein Mse7, wherein the DNA binding protein Mse7 contains an Avi-tag label.

[0008] Furthermore, the amino acid sequence of the protein Mse7 is as shown in SEQ ID NO.1, the amino acid sequence of the biotin ligase is as shown in SEQ ID NO.2, and the Avi-tag sequence is GLNDIFEAQKIEWHE.

[0009] Preferably, the SEQ ID NO.1: MATKIKFKYKGQDLEVDISKVKKVWKVGKMVSFTYDDNGKTGRGAVSEKDAPKELLNMIGKK;

[0010] Furthermore, the amino acid sequence of the DNA binding protein Mse7 connected with the Avi-tag tag sequence is: GLNDIFEAQKIEWHEMATKIKFKYKGQDLEVDISKVKKVWKVGKMVSFTYDDNGK TGRGAVSEKDAPKELLNMIGKK, and the gene encoded by it is shown in SEQ ID NO.4.

[0011] The SEQ ID NO.2: MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHI QTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQD RKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK.

[0012] Furthermore, the gene encoding biotin ligase is shown in SEQ ID NO.3.

[0013] The method for preparing the chromatography medium based on the DNA binding protein Mse7 of the present invention comprises the following steps:

[0014] (1) Avi-tag was added to the amino acid sequence of DNA binding protein Mse7 and constructed into PET28a vector together with biotin ligase;

[0015] (2) co-expressing the amino acid vector plasmid containing the target protein obtained in step (1) and the biotin ligase vector plasmid in a DE3 competent medium, activating and inducing, and centrifuging to remove the supernatant to obtain bacterial sludge;

[0016] (3) The protein supernatant obtained by centrifugation after the bacterial sludge is crushed is passed through an affinity chromatography heparin column and an ion column to obtain a pure target protein, which is then concentrated and dialyzed;

[0017] (4) The concentrated protein is incubated with streptavidin agarose gel.

[0018] Furthermore, the process of purifying the protein in step (3) is as follows: first, the collected protein supernatant is passed through a heparin column, then the collected sample after heparin is passed through an SP column, the collected sample after passing through the SP column is passed through a Q column, the sample after passing through the Q column is added to a dialysis bag and then placed in polyethylene glycol for concentration, and the concentrated sample is placed in a low-salt buffer for dialyzation.

[0019] Furthermore, the molecular weight cut-off of the dialysis bag is 3-5 KDa, and the polyethylene glycol is polyethylene glycol 20000.

[0020] Preferably, the dialysis bag has a molecular weight cut-off of 3 KDa.

[0021] Furthermore, in step (4), the agarose gel is first taken and the supernatant is removed, the agarose gel is washed, the protein solution is diluted and placed in a streptavidin agarose gel for incubation overnight, a washing buffer is added to the agarose gel, the agarose gel is resuspended, the supernatant is taken by centrifugation, and finally the precipitate is taken, which is the synthesized new chromatography filler.

[0022] Furthermore, the incubation temperature is 0-4°C and the duration is 16-20 hours.

[0023] Among them, the filler of the present invention is used in removing host nucleic acid residues in recombinant proteins.

[0024] Furthermore, the application process is: flowing the RNase inhibitor solution through the chromatography filler and receiving the flow-through liquid at the same time, and detecting the host Ct value with a qPCR instrument.

[0025] Furthermore, the qPCR primers are: 23S rRNA-F: 5'GTCTGGAAAGGCGCGCGA 3'; 23S rRNA-R: 5'GTGTCCCGCCCTACTCA3'.

[0026] The present invention combines the biotinylated DNA binding protein Mse7 with streptavidin agarose gel for the first time to form a new chromatography medium for application in the purification of recombinant proteins, adopting a flow-through mode, that is, the target protein flows through when loading, and the host DNA is adsorbed on the chromatography filler to achieve the purpose of purification. The existing Q chromatography filler method for removing host nucleic acids requires the preparation of two or more high and low salt buffers, and the sample needs to be pretreated before passing through the Q filler, and then the target protein solution can be obtained by high salt elution. Due to its strong adsorption effect, it may produce non-specific adsorption to the target protein, resulting in a reduction in the yield of the target protein. The DNA binding protein Mse7 invented in this patent is a new filler that is short in time, high in efficiency, simple and safe to operate, does not affect the yield of the enzyme, and can effectively remove the host DNA residue in the biological tool enzyme. Break through the quality bottleneck of recombinant proteins and improve product quality standards. So far, there is no method for removing recombinant protein host nucleic acid residues by combining DNA binding protein with streptavidin to prepare a purification filler.

[0027] The present invention is the first to closely combine the biotinylated Mse7 protein specificity with streptavidin agarose gel to form a new type of chromatography filler. The DNA binding protein is first biotinylated so that it can be firmly bound to streptavidin. On this basis, a new type of filler is prepared. The RI protein solution is then passed through the new filler, which does not adsorb the RI protein but only binds to the host nucleic acid in the RI protein solution. Thereby achieving the purpose of removing the host nucleic acid in the recombinant protein.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0029] 1. Using the DNA binding protein Mse7 prepared by the present invention as a novel chromatography medium for purification of biological tool enzymes can effectively remove host DNA and improve product quality. Compared with traditional methods, this technology has higher purification efficiency and recovery rate.

[0030] 2. The DNA-binding protein Mse7 of the present invention adopts a flow-through mode to remove host nucleic acids in recombinant proteins. It is easy to operate, does not require complicated sample pretreatment, and has no negative impact on the environment and experimenters. It is fast and efficient: compared with traditional methods, this technology has higher purification efficiency and recovery rate, and can greatly shorten the purification time. Secondly, it saves time costs: due to its simple operation, rapidity and high efficiency, this technology can save a lot of time costs, which is conducive to improving production efficiency. Thirdly, it can reduce host DNA: the use of this new chromatography medium can effectively remove host DNA and reduce its impact on product quality. Finally, it can improve product quality standards: by effectively reducing host DNA, this technology helps to improve the quality standards of biological tool enzymes and meet the needs of the high-end market. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The schematic diagram of the preparation principle and application of the new Mse7 filler;

[0032] Figure 2 Construction of an expression vector for the biotin ligase BirA inserted into the Pet28a multiple cloning site;

[0033] Figure 3 Construction of expression vector for inserting Mse7-Avi tag into PET28a multiple cloning site;

[0034] Figure 4 The gel map confirmation of the new filler of Mse7;

[0035] Figure 5 The effect of 2MNaCl on Mse7 binding to SA during the preparation of the new filler;

[0036] Figure 6 Flow chart of the new filler and Q filler for the example RNase inhibitor;

[0037] Figure 7 The graphs are as follows: the sample after RI passed through the nickel column passed through the Q matrix (filler) and the new filler respectively; the graphs are as follows: the sample after RI passed through the Q column passed through the Q filler and the new filler respectively;

[0038] Figure 8 This is the amplification curve of the sample after the RI nickel column in Example 1, the first time through the Q filler and the new filler;

[0039] Fig. 9 This is the amplification curve of the sample after the RIQ column in the embodiment, the first pass through the Q filler and the new filler;

[0040] Fig.10 This is the amplification curve of the sample after the RI nickel column in Example 1, the second pass through the Q filler and the new filler;

[0041] Fig.11This is the amplification curve of the sample after the RIQ column in the embodiment, the second pass through the Q filler and the new filler;

[0042] Fig.12 This is the amplification curve of the sample after the RI nickel column in Example 1, the third time through the Q filler and the new filler;

[0043] Fig.13 This is the amplification curve of the sample after the RIQ column in Example 1, the third pass through Q filler and the new filler. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0045] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0046] The English letters of the present invention are described as follows:

[0047] Flow: flow-through; Wash: washing solution; Elute: elution solution; SA: streptavidin.

[0048] Drugs and reagents:

[0049] Low salt buffer A: 20 mM NaPO4, 0.1 mM EDTA, 5% glycerol, pH 7.0 @ 25 °C;

[0050] High salt buffer B: 20 ​​mM NaPO4, 0.1 mM EDTA, 5% glycerol, 1 M NaCl pH 7.0 @ 25 °C;

[0051] Heparin affinity chromatography medium: Zhongke Senhui Microsphere Technology (Suzhou) Co., Ltd., catalog number: 16-0020-06;

[0052] SP packing SP Sanarose HP: Xiamen Sanji Technology Co., Ltd., item number: NSH00100;

[0053] Capto Q ion exchange chromatography medium: Cytiva, batch number: 17531610;

[0054] Nickel filler Ni-IDA Sanarose 6FF: Xiamen Sanji Technology Co., Ltd., catalog number: NIA01000;

[0055] Prepacked plastic empty column (ion exchange column, PrePack CM Q Purose 6HP): Jiaxing Qianchun Biotechnology Co., Ltd., catalog number: ZA20304-16;

[0056] The present invention adds nickel filler, heparin, Q filler and SP filler into the empty column to form nickel column, heparin column, Q column and SP column for experiment, wherein the anion column is Q column and the cation column is SP column.

[0057] Recombinant protein of ribonuclease inhibitor (RI): recombinant mouse RNase inhibitor, Jiangsu Best Biotechnology Co., Ltd., catalog number: EG20002S;

[0058] 4×SDS protein loading: Jiangsu Baishimei Biological Co., Ltd., batch number: 3234803241.

[0059] Example 1

[0060] like Figure 1 As shown, a method for removing host nucleic acid comprises the following steps:

[0061] Step 1: Construct vector

[0062] The protein sequence of biotin ligase (GenBank: AAN88945.1) was searched on the NCBI website. After codon optimization, the gene sequence (SEQ ID NO.3) was synthesized by a gene synthesis company and inserted into the Pet28a multiple cloning site to construct an expression vector (between the restriction sites NcoI and EcoRI). The expression vector model is as follows: Figure 2 Similarly, the protein sequences of Avi-tag (GenBank: QBQ88493.1) and Mse7 (GenBank: A4YEA2) were obtained from the NCBI website. After codon optimization, the Avi tag-Mse7 gene sequence (SEQ ID NO.4) was sent to a gene company for synthesis and inserted into the PET28a multiple cloning site to construct an expression vector (between the restriction sites NcoI and EcoRI). The expression vector model is as follows Figure 3 shown.

[0063] Step 2: Prepare bacterial mud

[0064] 1. Plate transformation steps:

[0065] Take 100ng Mse7 plasmid and 100ng bioligase plasmid, co-transform and add to 50μL competent BL21 (DE3), and place on ice for 30min; heat shock at 42℃ for 1min, and place on ice for 5min; add 500μL antibiotic-free culture medium, and activate at 37℃, 200rpm for 1h; take 100μL activation solution and apply to kanamycin resistance plate, invert and place at 37℃ for about 16h;

[0066] 2. Vial activation stage:

[0067] Select about 50 monoclonal colonies and inoculate them into 200 mL of culture medium, activate them in a 1000 mL shake flask, grow them for 3.5 h at 37°C and 200 rpm, and measure the OD 600 When UV is about 0.8, proceed to the next step;

[0068] 3. Large bottle expansion stage:

[0069] Prepare 3 bottles of 500 mL culture medium, inoculate each bottle with 50 mL activation solution (10% inoculation amount), expand the culture in a 2000 mL shake flask, 37°C, 200 rpm, grow for 3.5 hours, and measure OD 600 UV is around 0.8;

[0070] 4. Large bottle induction stage:

[0071] Add IPTG to each bottle at a final concentration of 1mM for induction, 30℃, 200rpm, and end the fermentation after 4h of induction. Collect the bacteria; centrifuge to collect the bacteria; fermentation volume is 1.5L; measure OD 600 UV 1.83; 9800g, 4℃ centrifugation for 10 min to collect bacteria; discard the supernatant to obtain bacterial sludge, weighing 9.8g.

[0072] 5. Sample pretreatment:

[0073] Take 7g of fermented bacterial sludge; add 70mL of lysis solution (20mM NaPO4 (pH7.0@25℃), 0.1mM EDTA, 5% glycerol), then add 70μL of protease inhibitor 100mM PMSF and 70μL of 1000×PI to resuspend; then crush at high pressure of 850bar for 10min until the bacterial solution is clear. Finally, centrifuge at 25000g, 4℃ for 30min, take the supernatant, and obtain the crude enzyme solution sample.

[0074] Step 3: Protein purification

[0075] A three-step purification scheme is used to purify the target protein. Heparin HP filler is combined with cationic SP filler for purification; finally, in order to reduce the host content of the protein itself, it is passed through anionic Q filler and the Flow is collected. The specific steps are as follows:

[0076] 1. The supernatant of crude enzyme solution is passed through a heparin column

[0077] (1) Equilibration: Run 3 column volumes with low salt buffer (20 mM NaPO4 (pH 7.0 @ 25 °C), 0.1 mM EDTA, 5% glycerol) until the conductivity and UV280 are stable, and set the flow rate to 2 ml / min;

[0078] (2) Sampling: 70 ml of the crude enzyme solution obtained by the above sample pretreatment is loaded through the sample pump pipeline, and the flow rate is set to 1 ml / min;

[0079] (3) Cleaning: Run 3CV with low-salt buffer. When the conductivity and UV280 baselines are stable, the cleaning is completed and the flow rate is set to 1 ml / min.

[0080] (4) Elution: Use pipelines A and B, the buffer of pipeline A1 is low-salt buffer A, and the buffer of pipeline B is high-salt buffer B (20 mM NaPO4 (pH 7.0 @ 25 ° C), 0.1 mM EDTA, 5% glycerol, 1 M NaCl), set the flow rate to 2 ml / min, 0%-100% high-salt buffer eluent, and elute for 10 column volumes.

[0081] (5) Collection: Record the number of each tube, record the collection volume, collect the Flow, Wash, Elute after the heparin column, add protein 4×SDS protein loading to run SDS-PAGE, observe whether there is the target band of Mse7, collect several tubes of Elute with the target band and mix them together for the next column purification.

[0082] 2. Collect the sample after heparinization and pass it through SP column

[0083] (1) Balance: same as heparin column;

[0084] (2) Sample loading: The sample conductivity of the ion packing material is kept within 10 mS / cm. The heparinized sample is diluted to the corresponding conductivity value with a low salt buffer (20 mM NaPO4 (pH 7.0 @ 25°C), 0.1 mM EDTA, 5% glycerol).

[0085] (3) Cleaning: Same as heparin column;

[0086] (4) Elution: same as heparin column;

[0087] (5) Collection: same as heparin column;

[0088] 3. Collect the samples after passing through the SP column and pass them through the Q column to reduce the host residues of the bound protein itself. At the same time, the Q column can also improve the protein purity.

[0089] The process of protein purification by the Q column is as follows:

[0090] (1) Balance: Same as SP column;

[0091] (2) Sample loading: The sample conductivity of the ion packing material should be kept within 10 mS / cm. Dilute the sample after SP with low-salt buffer to the corresponding conductivity value. Collect the flow for future use.

[0092] 4. Collect the samples after Q and concentrate them in polyethylene glycol 20000 to increase the concentration. The process is as follows:

[0093] Prepare a beaker, wash it, fill it with an appropriate amount of distilled water, and heat it to a slight heat. Cut a 3Kda molecular weight cutoff dialysis bag of appropriate length according to the sample volume, put on disposable gloves and soak the cut dialysis bag in water for 10 minutes. Clamp one end of the sealing film with a clamp, pour Watsons water into the dialysis bag, and there is no leakage in the dialysis bag. After confirming the sealing of the dialysis bag, pour out the solution, use a pipette to carefully absorb the protein sample and add it to the dialysis bag. Lay it flat on polyethylene glycol 20000 for concentration, remove the wet polyethylene glycol 20000 on the dialysis bag every half an hour, replace it with new polyethylene glycol 20000, and concentrate for about 4 hours.

[0094] 5. The concentrated sample is dialyzed in a low-salt buffer.

[0095] The concentrated sample was taken out and placed in a 50ml centrifuge tube without zymogen. A dialysis bag of appropriate length (dialysis bag molecular weight cutoff is 3KDa) was reassembled according to the above operation, and the sample in the tube was sucked into a new dialysis bag. The dialysis bag was placed horizontally in a low-salt dialysis solution (20mM NaPO4 (pH7.0@25℃), 0.1mM EDTA, 5% glycerol), the magnetic stirrer was turned on, and the dialysis solution was collected after dialysis for 2h to obtain the biotinylated Mse7 protein.

[0096] Step 4: Preparation of new filler:

[0097] The biotinylated Mse7 protein prepared in step 3 was combined with streptavidin agarose gel to prepare a new filler. All preparation steps used a filter cartridge box pipette tip and were performed on ice. The specific steps are as follows:

[0098] (1) Quantitative marking: Take a 15 ml sterilized centrifuge tube, add 600 ul of enzyme-free water, and mark the location of the 600 ul water; after marking, use a gun to remove the water. Take out Streptavidin Agarose (SA, Beyotime, Cat No. P2159-5ml) from the -20°C refrigerator and dissolve it on ice.

[0099] (2) Take agarose gel and remove the supernatant: Gently resuspend the streptavidin agarose gel to form a uniform gel suspension, take 600ul of Beyotime's streptavidin agarose gel (excluding the solution), place it in a 15ml centrifuge tube for later use. Centrifuge at 1000×g for 1min at 4℃ and discard the supernatant after centrifugation.

[0100] (3) Washing agarose gel: Add TBS (10 mM Tris, pH 7.4) to a final volume of about 4 ml, gently resuspend the streptavidin agarose gel, centrifuge at 1000 × g at 4°C for 5 min, carefully remove the supernatant without aspirating the gel, complete one washing step, and then wash twice more.

[0101] (4) Protein adsorption: Take 2.4 mg of biotinylated Mse7 protein, add 2 ml of Washing Buffer II (10 mM Tris (pH 7.4), 0.05% Tween-20, PH 7.4) to dilute according to the volume of 2 ml of protein taken, mix well and take 100 ul as the control before incubation. Add the remaining samples to the agarose gel in the above step, resuspend and place on a rotating mixer, incubate at 4 ° C for 16 hours overnight, centrifuge at 1000g at 4 ° C for 5 minutes the next day, take 100 ul of the supernatant and apply it to the gel to ensure that the adsorption amount of DNA binding protein is overloaded.

[0102] (5) Washing: Washing Buffer I (10mM Tris (pH7.4), 0.05% Tween-20, 1M NaCl, pH7.4) contains 1M NaCl. High salt can remove the host content in the newly prepared filler. Take 4ml of Washing Buffer I and add it to the agarose gel, and gently resuspend the agarose gel. Centrifuge at 1000g, 4℃ for 5 minutes, remove the supernatant, and take the precipitate, which is the newly synthesized chromatography filler (Mse7 filler). The new filler is washed twice.

[0103] Finally, wash once more with 4 ml of Washing Buffer II, centrifuge, and take 3.9 ml of supernatant. After each wash, take 100 ul of supernatant and apply gel. Use the remaining 100 ul of the new filler to mix with Washing Buffer II, and take the remaining precipitate of the new filler and apply gel to confirm that the biotinylated DNA binding protein is bound to the streptavidin gel. Figure 4 As shown in the protein gel, lanes 1 and 8 are protein markers; lane 2 is Mse7 protein, i.e., the solution before incubation of Mse7 with SA; lane 3 is the supernatant after incubation of Mse7 with SA; lanes 4, 5, and 6 are the supernatants after washing the newly prepared filler with Washing Buffer; and lane 7 is the newly prepared filler. It can be seen from lane 3 that there is still a large amount of Mse7 protein in the supernatant after incubation of Mse7 with SA, indicating that the protein binding amount has reached saturation. It can be seen from lane 3 that there is no target protein in the supernatant after washing the newly prepared filler with Washing Buffer, indicating that the Mse7 protein is not easy to fall off from SA. It can be seen from lane 7 that Mse7 has specifically bound to SA, indicating that Mse7 is a biotinylated protein.

[0104] Example 2

[0105] The method of Example 1 was used, except that in step 4, 1M NaCl in the washing process was replaced with 2M NaCl in the preparation of the new filler, and the other processes remained unchanged. Figure 5 As shown, lane 1 is the newly prepared filler, lane 2 is the protein marker, and lane 3 is the supernatant after incubation of Mse7 and SA. It can be seen from lane 1 that Mse7 and SA cannot bind, indicating that 2M NaCl, that is, high salt solution, will affect the binding of DNA binding protein to streptavidin agarose gel.

[0106] Example 3

[0107] RI flows through new filler and Q filler to detect host Ct value

[0108] Protein RNase inhibitor (RI) was used to compare the ability of newly prepared fillers and anionic fillers to remove host. The reasons for using RI are: first, RI has no ability to degrade, cut, or eliminate nucleic acid fragments; second, RI has a high protein yield; and third, the purification process contains anionic columns to remove host and impurities.

[0109] like Figure 6 As shown, RI first passes through the nickel column, and a small portion of the sample after the nickel column is passed through 45ul of the newly prepared filler and 45ul of the Q filler in Example 1 to determine the ability of the two fillers to remove the host under high host conditions. The remaining sample after the nickel column passes through the Q column, and a small portion of the sample after the Q column is passed through 45ul of the newly prepared filler and 45ul of the Q filler to determine the ability of the two fillers to remove the host under low host conditions.

[0110] The specific operations are as follows:

[0111] (1) RI new filler: Take a 1.5ml high temperature sterilized EP tube, add 45ul enzyme-free raw water, mark the position of 45ul water, and remove the water with a gun after marking.

[0112] (2) The newly prepared Mse7 filler precipitate filler in Example 1 was divided into 6 portions, each portion containing 45u1 of filler, and 3 parallel samples were made to reduce errors. Excess Washing Buffer II was removed by centrifugation, and 150ug and 300ug of the sample after RI passed through the nickel column, and 150ug and 300ug of the sample after RI passed through the Q column were taken into 1.5ml EP tubes, and then 45ul of the newly prepared Mse7 filler was added, respectively, and incubated on a shaker at 4℃, 230rpm for 2h; 1000g, 4℃ centrifuged for 4min to collect the supernatant. Two different ratios of sample to filler were designed, sample: filler = 1:2 (150ug: 45u1, w / v, ug / ul), sample: filler = 2:1 (300ug: 45u1, w / v, ug / ul), in order to eliminate the influence of different sample to filler ratios on host removal.

[0113] (3) RI over Q filler: Take a 1.5 ml high temperature sterilized EP tube, add 45 ul enzyme-free raw water, mark the position of the 45 ul water, and remove the water with a gun after marking.

[0114] (4) Take 45ul of 6 portions of Q filler (anion filler), centrifuge and remove the supernatant. Add 1ml of RI low-salt buffer (20mMNaH2PO4-NaOH, 0.1mM EDTA, 1mM DTT, 5% glycerol (pH6.0@25℃)), vortex gently to mix, then centrifuge at 1000×g for 1min at 4℃, wash twice to completely remove 20% ethanol in the filler. Take 150ug and 300ug of the sample after RI passed through the nickel column and 150ug and 300ug of the sample after RI passed through the Q column and pass through 45ul of Q filler, where two different ratios of sample and filler are designed, sample: filler = 1:2 (150ug: 45u1, w / v, ug / ul), sample: filler = 2:1 (300ug: 45u1, w / v, ug / ul). Incubate on a shaker for 2h, 4℃, 230rpm; centrifuge at 1000g, 4℃ for 1min, and remove the supernatant. Add 1 ml of RI low-salt buffer (20 mM NaH2PO4-NaOH, 0.1 mM EDTA, 1 mM DTT, 5% glycerol (pH 6.0 @ 25 ° C)) to wash, centrifuge and remove the supernatant. Then add 150 ul of 40% RI high-salt buffer (20 mM NaH2PO4-NaOH, 1 M NaCl, 0.1 mM EDTA, 1 mM DTT, 5% glycerol (pH 6.0 @ 25 ° C)) and 225 ul of 60% RI low-salt buffer to elute and incubate for 0.5 h. After elution and incubation, centrifuge at 1000 g, 4 ° C, 4 min, and collect the supernatant.

[0115] (5) The supernatant collected in steps (2) and (4) was subjected to fluorescence quantitative PCR detection. The results are shown in Table 1:

[0116] Table 1 Fluorescence quantitative PCR detection results

[0117]

[0118]

[0119] According to the above table and attached Figures 7 to 13It can be seen that. RI sample 150ug and filler 45u1, as a sample to filler ratio of 1:2; RI sample 300ug and filler 45u1, as a sample to filler ratio of 2:1. There is no significant difference in host removal with different sample to filler ratios. The newly prepared filler prepared by combining Mse7 with streptavidin agarose gel has the ability to remove the host in the sample after RI passes through the nickel column in Example RI, that is, under the background of high background host content, but the difference is significant compared with the Q filler. In the sample after RI passes through the Q column in Example RI, that is, under the background of low background host content, the newly prepared filler has the ability to remove the host, and the effect is basically the same as that of the anionic Q filler. Therefore, the DNA binding protein Mse7 has the ability to remove host nucleic acid residues in recombinant proteins. In summary, the present invention can achieve the purpose of removing host DNA through a one-step method, that is, the RI protein flow passes through the new filler, so that the residual host DNA of the finished recombinant protein is further reduced, the time cost is greatly reduced, multi-step operation is avoided, and the quality of the tool enzyme product is improved.

Claims

1. A chromatography medium based on DNA binding protein Mse7, characterized in that: The chromatography medium comprises biotin ligase, streptavidin agarose gel and DNA binding protein Mse7, wherein the DNA binding protein Mse7 contains an Avi-tag label.

2. The chromatography medium based on the DNA binding protein Mse7 according to claim 1, characterized in that: The amino acid sequence of the protein Mse7 is shown in SEQ ID NO.1, the amino acid sequence of the biotin ligase is shown in SEQ ID NO.2, and the Avi-tag sequence is GLNDIFEAQKIEWHE.

3. A method for preparing a chromatography medium based on the DNA binding protein Mse7 according to claim 1, characterized in that: The steps include: (1) An Avi-tag was added to the amino acid sequence of the DNA binding protein Mse7, and its encoding gene and the biotin ligase encoding gene were constructed into the PET28a vector respectively; (2) co-expressing the target protein encoding gene vector plasmid obtained in step (1) and the biotin ligase encoding gene vector plasmid in competent Escherichia coli, activating and inducing, and centrifuging to remove the supernatant to obtain bacterial sludge; (3) The protein supernatant obtained by centrifugation after the bacterial sludge is crushed is passed through an affinity chromatography heparin column and an ion column to obtain a pure target protein, which is then concentrated and dialyzed; (4) The concentrated protein is incubated with streptavidin agarose gel.

4. The method for preparing a chromatography medium based on the DNA binding protein Mse7 according to claim 3, characterized in that: The process of purifying the protein in step (3) is as follows: first, the collected protein supernatant is passed through a heparin column, then the sample after the heparin is collected is passed through an SP column, the sample after the SP column is collected is passed through a Q column, the sample after the Q column is added to a dialysis bag and then placed in polyethylene glycol for concentration, and the concentrated sample is placed in a low-salt buffer for dialyzation.

5. The method for preparing a chromatography medium based on the DNA binding protein Mse7 according to claim 4, characterized in that: The molecular weight cut-off of the dialysis bag is 3-5 KDa, and the polyethylene glycol is polyethylene glycol 20000.

6. The method for preparing a chromatography medium based on the DNA binding protein Mse7 according to claim 3, characterized in that: The step (4) first takes streptavidin agarose gel and removes the supernatant, washes the agarose gel, dilutes the protein solution and places it in the streptavidin agarose gel for incubation overnight, takes a washing buffer and adds it to the agarose gel, resuspends the agarose gel, centrifuges and removes the supernatant, and finally takes the precipitate to obtain the synthesized new chromatography filler.

7. The method for preparing a chromatography medium based on the DNA binding protein Mse7 according to claim 6, characterized in that: In step (4), the incubation temperature is 0-4°C and the incubation time is 16-20 hours.

8. Use of the chromatography medium based on the DNA binding protein Mse7 according to claim 1 in removing host nucleic acid residues in recombinant proteins.

9. The use according to claim 8, characterized in that: The application process is: let the RNase inhibitor solution flow through the filler and receive the flow-through liquid at the same time, and then detect the host Ct value with a qPCR instrument.

10. The use according to claim 9, characterized in that: The qPCR primers are: 23S rRNA-F: 5'GTCTGGAAAGGCGCGCGA3'; 23S rRNA-R: 5'GTGTCCCGCCCTACTCA3'.

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