Chromatographic process for removing host nucleic acid residues in Bst DNA polymerase and Bst DNA polymerase

Abstract

According to the chromatographic process for removing the host nucleic acid residues in the Bst DNA polymerase and the Bst DNA polymerase, the crude enzyme containing the Bst DNA polymerase is subjected to enzymolysis and three-step chromatography including affinity chromatography, anion exchange chromatography and hydrophobic chromatography, the nucleic acid residues in the crude enzyme can be removed, the prepared Bst DNA polymerase is free of nucleic acid residues, and tests prove that the host nucleic acid residues in the Bst DNA polymerase can be effectively removed. The purity of the product Bst DNA polymerase obtained through the purification method is higher than 95%, host DNA residues are avoided, the product Bst DNA polymerase can be well applied to LAMP reaction, and nonspecificity caused by HCD rising is reduced.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a chromatography process for removing host nucleic acid residues in Bst DNA polymerase and Bst DNA polymerase. Background Art

[0002] Medical products such as molecular diagnosis are now widely known to people, especially molecular diagnostic products based on nucleic acid amplification technology. With the characteristics of high sensitivity, strong specificity, fast speed, short diagnostic window period and qualitative / quantitative detection, they are widely used in hepatitis, pulmonary infectious diseases, eugenics, genetic disease genes and tumors, etc., filling the detection gap in the immune detection window period. For example, in the application of LAMP isothermal amplification technology, nonspecific amplification directly produces false positives, and due to the large amount of products in the LAMP reaction, once nonspecific amplification occurs, it is easy to contaminate the laboratory environment, resulting in continuous false positive results, which seriously affects the diagnostic test results; the residual nuclease of the raw material is one of the key factors affecting nonspecific amplification. Under high-sensitivity detection methods, even trace amounts of nucleic acid residues may reduce the performance of diagnostic reagents, thereby causing many technical problems. Bst DNA polymerase is the core enzyme raw material in LAMP isothermal amplification technology, and the HCD content needs to be strictly controlled; there are currently some purification processes for Bst DNA polymerase, but none of them mention the removal of HCD. Summary of the invention

[0003] In view of this, it is necessary to provide a chromatography process for removing host nucleic acid residues in Bst DNA polymerase in view of the current technical defects.

[0004] To solve the above problems, this application adopts the following technical solutions:

[0005] One of the purposes of the present application is a chromatography process for removing host nucleic acid residues in Bst DNA polymerase, characterized in that it comprises the following steps:

[0006] Performing precipitation treatment on the crude enzyme solution to obtain a crude enzyme supernatant;

[0007] treating the crude enzyme supernatant with nuclease to obtain an enzymatic hydrolyzate;

[0008] performing affinity chromatography on the enzymatic hydrolysate to obtain a first eluted sample;

[0009] performing anion exchange chromatography on the first eluted sample to obtain a second eluted sample;

[0010] performing hydrophobic chromatography on the second eluted sample to obtain a third eluted sample;

[0011] The third eluted sample is concentrated and ultrafiltered to obtain a pure Bst DNA polymerase product without residual host nucleic acid.

[0012] In some of the embodiments, the step of precipitating the crude enzyme solution to obtain the crude enzyme supernatant specifically includes the following steps: crushing the fermented bacterial sludge of the Bst DNA polymerase, adding a flocculant or a water-soluble cationic polymer with a final volume concentration of 0.1%-1%, standing for solid-liquid separation, collecting the supernatant, and obtaining the crude enzyme supernatant.

[0013] In some of the embodiments, the step of treating the crude enzyme supernatant with nuclease to obtain an enzymatic solution specifically includes the following steps: adjusting the pH of the crude enzyme supernatant to 8.0-9.0, adding 1-5mM MgCl2 and 200U / g bacteria-1000U / g bacteria nuclease to the supernatant, and reacting at 37°C for 1h.

[0014] In some embodiments, the step of performing affinity chromatography on the enzymatic hydrolysate to obtain a first eluted sample specifically includes the following steps:

[0015] Equilibrium affinity chromatography column;

[0016] After adding 300mM-500mM NaCl to the enzymatic hydrolysate, the sample is loaded onto the equilibrated affinity chromatography column;

[0017] Carrying out a washing treatment on the affinity chromatography column after loading the sample;

[0018] The affinity chromatography column after the washing treatment is eluted with a first elution buffer to obtain the first eluted sample.

[0019] In some embodiments, the affinity chromatography column is a Ni column affinity chromatography, and the affinity chromatography medium is selected from any one of NiSepharose HP, Ni Sepha rose 6FF, TALON Superflow, and Ni Sepharose excel;

[0020] The pH of the equilibration buffer used to balance the affinity chromatography column is 7.0-8.0, and includes 10mM-50mM Tris HCl, 300mM-500mM NaCl, and 10mM-50mM imidazole;

[0021] The pH of the washing buffer used for washing the affinity chromatography column after loading is 7.0-8.0, and includes 10mM-50mM Tris HCl and 500mM-2M NaCl;

[0022] The first elution buffer has a pH of 7.0 to 8.0 and includes 10 mM to 50 mM Tris HCl, 50 mM to 200 mM NaCl, 100 mM to 200 mM imidazole

[0023] In some embodiments, the step of subjecting the first eluted sample to anion exchange chromatography to obtain a second eluted sample specifically includes the following steps:

[0024] Equilibrating anion exchange chromatography columns;

[0025] directly loading the first eluted sample onto the equilibrated anion exchange chromatography column;

[0026] The loaded anion exchange chromatography column is eluted with a second elution buffer to obtain the second eluted sample.

[0027] In some embodiments, the chromatography medium used is selected from any one of Q Sepharose FF, Q Sepharose 4FF, Q Sepharose XL, Q Sepharose HP, Q Sepharose Big Beads, Capto Q, DEAE Sepharose FF, DEAE Sepharose CL 6B, DEAE Sephacel, Capto DEAE, and Anx Sepharose 4FF;

[0028] The second elution buffer has a pH of 7.0 to 8.0 and includes 10 mM to 50 mM Tris HCl and 150 mM to 250 mM NaCl;

[0029] The pH of the balancing buffer for balancing the anion exchange chromatography column is 7.0-8.0, and includes 10mM-50mM Tris HCl and 50mM-250mM NaCl.

[0030] In some embodiments, the step of performing hydrophobic chromatography on the second eluted sample to obtain a third eluted sample specifically includes the following steps:

[0031] Equilibrating a hydrophobic chromatography column;

[0032] After adding 0.8M to 1.2M ammonium sulfate solid to the first elution buffer to make the final concentration of 0.8-1.2M ammonium sulfate, the sample is loaded onto the equilibrated hydrophobic chromatography column;

[0033] The loaded hydrophobic chromatography column is eluted with a third elution buffer to obtain the third elution sample.

[0034] In some embodiments, the chromatography medium used is selected from any one of Capto Phenyl FF, Phenyl Sepharose 6 Fast Flow, Capto Phenyl ImpRes, and Phenyl Sepharose High Performance; the pH of the third elution buffer is 7.0-8.0, and includes 10mM-50mM Tris HCl, 0.1M-0.5M ammonium sulfate; the pH of the balancing buffer for balancing the hydrophobic ion exchange chromatography column is 7.0-8.0, and includes 10mM-50mM Tris HCl, 0.8M-1M ammonium sulfate.

[0035] The second purpose of the present application is to provide a Bst DNA polymerase, which is prepared by any of the chromatography processes for removing host nucleic acid residues in the Bst DNA polymerase.

[0036] This application adopts the above technical solution, and its beneficial effects are as follows:

[0037] The present application provides a chromatography process for removing host nucleic acid residues in Bst DNA polymerase and a Bst DNA polymerase, wherein a crude enzyme solution containing Bst DNA polymerase is subjected to enzymatic hydrolysis and three-step chromatography, including affinity chromatography, anion exchange chromatography and hydrophobic chromatography, to remove nucleic acid residues in the crude enzyme solution, so that the prepared Bst DNA polymerase has no nucleic acid residues. It has been verified by experiments that the purity of the product Bst DNA polymerase obtained by the purification method is higher than 95%, there is no host DNA residue, and it can be well used in LAMP reaction to reduce the nonspecificity caused by increased HCD. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0039] Figure 1 This is an SDS PAGE image of the one-step affinity chromatography process of the enzymatic solution containing Bst DNA polymerase in Example 3;

[0040] Figure 2 This is an SDS PAGE image of the affinity chromatography elution collection fraction in Example 4 after two-step anion exchange chromatography;

[0041] Figure 3 This is an SDS PAGE image of the collected components eluted by anion exchange chromatography in Example 5 after three-step hydrophobic chromatography;

[0042] Figure 4 This is an SDS PAGE image of the finished Bst DNA polymerase obtained in Example 6;

[0043] Figure 5 The quality test result of the finished Bst DNA polymerase obtained in Example 6;

[0044] Figure 6 This is the Bst DNA polymerase activity stability test result obtained in Example 7. DETAILED DESCRIPTION

[0045] Embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0046] In the description of the present application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0047] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0048] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0049] One embodiment of the present application provides a chromatography process for removing HCD, which can obtain Bst DNA polymerase without HCD residue. The chromatography process for removing HCD comprises the following steps:

[0050] Step S1: performing precipitation treatment on the crude enzyme solution to obtain a crude enzyme supernatant.

[0051] In this embodiment, before the step of precipitating the crude enzyme solution to obtain the crude enzyme supernatant, the step of preparing the crude enzyme solution is also included, which specifically includes the following steps: crushing the fermented bacterial sludge of the Bst DNA polymerase, adding a flocculant or a water-soluble cationic polymer with a final volume concentration of 0.1%-1%, standing for solid-liquid separation, collecting the supernatant, and obtaining the crude enzyme supernatant.

[0052] In one specific example, the water-soluble cationic polymer is a flocculant.

[0053] Among them, the step of crushing the fermented bacterial sludge producing Bst DNA polymerase includes: adding the crushing buffer to the fermented bacterial sludge and mixing it evenly according to the ratio of the mass of the fermented bacterial sludge to the volume of the crushing buffer of 1:10, and crushing it under high pressure to obtain a crushing liquid. Specifically, the pH of the crushing buffer is 8.0, and it includes 20mM Tris-HCl, 200mM KCl, 5% (volume percentage) glycerol, and 0.1% (volume percentage) Tween 20. The pressure of high-pressure crushing is 800bar, and the crushing time is 10min.

[0054] Among them, in the standing step, the time is 2h to 20h, and the standing temperature is 4°C.

[0055] The solid-liquid separation method is centrifugation, specifically centrifugation at 4°C and 10,000 rpm for 30 min. It should be noted that the solid-liquid separation method is not limited to centrifugation, and may be other solid-liquid separation methods, such as filtration.

[0056] It can be understood that by adding flocculants or water-soluble cationic polymers, most of the host's own impurities in the crude enzyme solution can be removed, thereby improving the purity and activity of Bst DNA polymerase.

[0057] Step S2: treating the crude enzyme supernatant with nuclease to obtain an enzymatic hydrolyzate.

[0058] In this embodiment, the pH of the supernatant is adjusted to 8.0-9.0, 1-5 mM MgCl2 and 500U / g-2000U / g of bacterial nuclease are added to the supernatant, and the supernatant is incubated at 37°C for 1 hour and then filtered. A large amount of residual host nucleic acid in the crude enzyme solution can be removed by nuclease hydrolysis.

[0059] It can be understood that affinity chromatography can remove most of the host's own impurities in the enzymatic solution, thereby improving the purity and activity of Bst DNA polymerase.

[0060] Step S3: performing affinity chromatography on the above enzymatic hydrolysate to obtain a first eluted sample.

[0061] In some of the embodiments, the Bst DNA polymerase carries a His tag, and the affinity chromatography on the enzymatic hydrolysate is performed by Ni column affinity chromatography.

[0062] Wherein, the affinity chromatography medium is selected from any one of Ni Sepharose HP, Ni Sepharose 6FF, TALON Superflow, and Ni Sepharose excel.

[0063] In some embodiments, the step of performing affinity chromatography on the enzymatic hydrolysate comprises:

[0064] Step S31: balancing the affinity chromatography column;

[0065] The affinity chromatography column is balanced with a balancing buffer. Further, the pH of the balancing buffer used for the balancing affinity chromatography column is 7.0 to 8.0, and includes 10mM to 50mM Tris-HCl, 300mM to 500mM NaCl, and 10mM to 50mM imidazole. Further, the affinity chromatography column is balanced with a 5CV to 10CV (CV is the column volume, which is the same meaning below and will not be repeated) balancing buffer.

[0066] Before the step of balancing the affinity chromatography column, the method further includes the following steps: pretreating the chromatography medium with 2CV to 3CV 500mM imidazole, and then washing the affinity chromatography medium with 5CV to 10CV deionized water until the pH is neutral.

[0067] Step S32: After adding 300mM-500mM NaCl to the enzymatic hydrolysate, the hydrolysate is loaded onto the equilibrated affinity chromatography column.

[0068] The sample is loaded after adding 300mM NaCl and 30mM imidazole to the enzymatic solution containing Bst DNA polymerase, which is more conducive to the binding of Bst DNA in the enzymatic solution to the chromatographic medium and the flow through of impurities, thereby improving the purity of Bst DNA polymerase. The column volume of the affinity column is 25mL, and the flow rate of loading is 6mL / min to 10mL / min.

[0069] Step S33: washing the affinity chromatography column after loading the sample.

[0070] The washing treatment can remove the residual host nucleic acid and the weakly bound impurity proteins on the chromatography column, improve the purity of BstDNA polymerase, and avoid the influence of impurity proteins on the activity of BstDNA polymerase, thus ensuring that BstDNA polymerase has high activity.

[0071] Step S34: using a first elution buffer to elute the affinity chromatography column after the impurity washing treatment to obtain a first elution sample.

[0072] Wherein, the affinity chromatography column is a Ni column affinity chromatography, and the affinity chromatography medium is selected from any one of Ni SepharoseHP, NiSepha rose 6FF, TALON Superflow, and Ni Sepharose excel;

[0073] The pH of the equilibration buffer used to balance the affinity chromatography column is 7.0-8.0, and includes 10mM-50mM Tris HCl, 300mM-500mM NaCl, and 10mM-50mM imidazole;

[0074] The pH of the washing buffer used for washing the affinity chromatography column after loading is 7.0-8.0, and includes 10mM-50mM Tris HCl and 500mM-2M NaCl;

[0075] The first elution buffer has a pH of 7.0-8.0 and includes 10 mM-50 mM Tris HCl, 50 mM-200 mM NaCl, and 100 mM-200 mM imidazole.

[0076] By using the first elution buffer, the Bst DNA polymerase connected to the chromatography column can be eluted more thoroughly, and the elution of impurities such as foreign proteins can be avoided, thereby improving the purity and activity of the Bst DNA polymerase.

[0077] Step S4: performing anion exchange chromatography on the first eluate to obtain a second elution sample.

[0078] In this embodiment, the step of performing anion exchange chromatography on the first eluted sample to obtain the second eluted sample specifically includes the following steps:

[0079] Step S41: equilibrate the anion exchange chromatography column.

[0080] Furthermore, the pH of the equilibration buffer used to equilibrate the anion exchange column is 7.0-8.0, and includes 10 mM-50 mM Tris-HCl. Furthermore, the anion exchange column is equilibrated with 5CV-10C equilibration buffer.

[0081] Before the step of balancing the anion exchange column, the following steps are included: washing the anion chromatography medium with 2CV to 3CV 2M NaCl and 2CV to 3CV 0.5M NaOH, and then washing the anion chromatography medium with 5CV to 10CV deionized water until the pH is neutral.

[0082] Step S42: directly loading the first eluted sample onto the equilibrated anion exchange chromatography column.

[0083] It can be understood that the conductivity of the first eluent is below 15 mS / cm, which is more conducive to the binding of the Bst DNA polymerase in the first eluent to the chromatography medium, thereby improving the yield of the Bst DNA polymerase.

[0084] The column volume of the anion exchange column is 25 mL, and the sample loading flow rate is 6 mL / min to 10 mL / min.

[0085] Step S43: using a second elution buffer to elute the loaded anion exchange chromatography column to obtain the second eluted sample.

[0086] It can be understood that DNA carries a large amount of negative charge, which can be removed by adsorption of anion exchangers. Anion exchange chromatography can remove residual host nucleic acids, and can also remove impurities such as residual proteins in the affinity chromatography eluate, further improving the purity and activity of Bst DNA polymerase.

[0087] The pH of the washing buffer used for washing the anion exchange column after loading is 7.0-8.0, and includes 10mM-50mM Tris-HCl and 100mM-200mM NaCl. Further, the washing buffer is used for washing the anion exchange column after loading with 5CV-10CV of washing buffer.

[0088] The washing treatment can remove weakly bound impurity proteins on the chromatography column, improve the purity of Bst DNA polymerase, and avoid the influence of impurity proteins on the activity of Bst DNA polymerase, thereby ensuring that Bst DNA polymerase has high activity.

[0089] The anion exchange column after the washing treatment is eluted with a second elution buffer to obtain a second elution sample. The second elution buffer has a pH of 7.0 to 8.0 and includes 10 mM to 50 mM Tris-HCl and 150 mM to 250 mM NaCl.

[0090] The use of the second elution buffer can not only more thoroughly elute the Bst DNA polymerase bound to the chromatography column, but also avoid the elution of impurities such as foreign proteins, thereby improving the purity and activity of the Bst DNA polymerase.

[0091] The anion exchange column after the washing treatment is eluted using 5CV to 10CV of the second elution buffer.

[0092] Step S5: performing hydrophobic chromatography on the second eluted sample to obtain a third eluted sample.

[0093] The Bst DNA polymerase can be further purified by hydrophobic chromatography to obtain a Bst DNA polymerase with higher activity and greater stability.

[0094] In some embodiments, in the step of performing hydrophobic chromatography on the second eluted sample, the chromatography medium used is selected from at least one of Phenyl Sepharose 6FF, Phenyl Sepharose HP, Butyl Sepharose 4FF, Butyl Sepharose 6FF, Butyl Sepharose HP, and Octyl Sepharose 4FF.

[0095] The pH of the equilibration buffer used for the hydrophobic chromatography is 7.0-8.0, and includes 10mM-50mM Tris-HCl and 0.5M-1M ammonium sulfate. Furthermore, the hydrophobic chromatography column is equilibrated with 5CV-10CV of the equilibration buffer.

[0096] Furthermore, before the step of balancing the hydrophobic chromatography column, the following steps are included: washing the hydrophobic chromatography medium with 2CV to 3CV of deionized water and 2CV to 3CV of 0.5M NaOH, and then washing the hydrophobic chromatography medium with 5CV to 10CV of deionized water until the pH is neutral.

[0097] The elution buffer used in the hydrophobic medium has a pH of 7.0 to 8.0 and includes 10 mM to 50 mM Tris-HCl and 0.1 M to 1 M ammonium sulfate, and the elution column volume is 5 CV to 10 CV.

[0098] Step S6: The third eluted sample is concentrated and ultrafiltered to obtain a pure BstDNA polymerase without host nucleic acid residues.

[0099] In the above-mentioned chromatography process for removing HCD, the crude enzyme solution containing Bst DNA polymerase is subjected to enzymatic hydrolysis and three-step chromatography, including affinity chromatography, anion exchange chromatography and hydrophobic chromatography, which can remove the host nucleic acid residues and various impurities in the crude enzyme solution, so that the prepared Bst DNA polymerase has trace amounts of HCD.

[0100] Further, the HCD removal chromatography process provided by the present application, after enzymatic hydrolysis and screening and combination of various chromatography fillers, obtains the purification process conditions of pre-treatment (i.e., acquisition of crude enzyme solution and nuclease hydrolysis), three-step chromatography and post-treatment (i.e., ultrafiltration and preservation), the pre-treatment can solve the problem of difficulty in filtering after bacterial mud crushing and residual host nucleic acid and more impurities in crude enzyme solution, and this step is easy to scale production; the three-step chromatography process includes affinity chromatography, anion exchange chromatography and hydrophobic chromatography, the process connection is easy, and it is easy to linearly amplify; and then after post-treatment, high-quality and highly stable Bst DNA polymerase can be output. The above-mentioned preparation method successfully prepared Bst DNA polymerase with low host DNA residue and enzyme purity up to 95%, with high purity and no RNase and Nickase pollution. The above-mentioned HCD removal chromatography process has the advantages of simple operation method, high yield, less HCD residue, high protein purity, high enzyme activity, and easy to amplify production. The results of property evaluation compared with mainstream products on the market show that the Bst DNA polymerase purified by the present invention has faster amplification speed, better sample compatibility, and better stability and specificity.

[0101] The present application also provides a Bst DNA polymerase, which is prepared by the above-mentioned chromatography process for removing HCD.

[0102] The above Bst DNA polymerase has high purity, no RNase and Nickase contamination, low host DNA residue, and better reproducibility, thermal stability and freeze-thaw stability.

[0103] The following is the specific embodiment section.

[0104] Unless otherwise specified, the reagents and instruments used in the examples are all conventionally selected in the art. The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions, such as the conditions described in the literature, books, or the methods recommended by the kit manufacturer. The reagents used in the examples are all commercially available.

[0105] Unless otherwise specified, in the following examples, the chromatography process was performed on an AKTA protein purifier.

[0106] Example 1: Collection of Bst DNA polymerase engineered bacterial sludge

[0107] The recombinant Escherichia coli expressing Bst DNA polymerase with a 6-His tag was revived and induced to ferment, and the fermentation liquid was collected by centrifugation at 10000 rpm for 30 min at 4°C.

[0108] Example 2: Preparation of Bst DNA polymerase digestion solution

[0109] Weigh 50g of the fermentation sludge collected in Example 1, add 500mL of pre-cooled crushing buffer (20mM Tris-HCl, 200mM NaCl, 5% (volume percentage) glycerol, 0.1% (volume percentage) Tween 20, pH 7.5) at a mass volume ratio of 1:10, mix well, and crush at 800bar for 30min to obtain a crushing liquid. At room temperature, a precipitant with a final volume concentration of 0.5% was added to the crushing liquid, and the supernatant was collected by centrifugation at 4°C for 30min after standing at 4°C for 16h. The supernatant is a crude enzyme solution containing Bst DNA polymerase, and the pH of the supernatant is adjusted to 8.0-9.0, and 2mM MgCl2 and 1000U / g bacterial nuclease are added to the supernatant, and the supernatant is collected by centrifugation at 4°C for 30min after acting at 37°C for 1h. The supernatant is the enzymatic hydrolyzate containing BstDNA polymerase. After adding 300mM KCl and 30mM imidazole, the supernatant is filtered through a 0.45μm filter membrane and can be used for further separation and purification.

[0110] Example 3: Affinity chromatography of Bst DNA polymerase

[0111] Take 25g of affinity chromatography medium Ni Sepharose HP and load it on XK 16 / 20, use 2-3CV 500mM imidazole to pretreat the chromatography medium, wash with deionized water until pH is neutral, and then use 5-10CV equilibration buffer (20mM Tris-HCl, 500mM NaCl, 30mM imidazole, 5% glycerol and 0.1% Tween 20, pH 7.5) to balance the chromatography column, add 500mM NaCl and 30mM imidazole at a final concentration to the enzymatic hydrolyzate, i.e., the components collected in Example 2, and filter after complete dissolution, and load the sample onto the equilibrated affinity chromatography column. After the loading is completed, use 5CV equilibration buffer to wash away the residual impurities on the column material, and use 5-10CV washing buffer (20mM Tris-HCl, 2M NaCl, 30mM imidazole, 5% glycerol and 0.1% Tween 20, pH 7.5) Wash away the weakly bound impurities on the chromatography column, use 5-10CV elution buffer (20mM Tris-HCl, 100mM NaCl, 100mM imidazole, 5% glycerol and 0.1% Tween 20, pH 7.5) to elute the target protein and collect it, then use 3-5CV washing buffer (10mM Tris-HCl, 500mM imidazole, 5% glycerol and 0.1% Tween 20, pH 7.5) to wash away the strongly bound proteins on the column material, and finally use deionized water to wash the buffer remaining on the column material, and finally store the column material in 20% ethanol as described in Example 3. The collected eluate containing the target protein was subjected to SDS-PAGE gel electrophoresis, and the detection results are as follows: Figure 1 As shown. Figure 1 It can be seen that most of the host's own impurities have been removed after one-step affinity chromatography. The test results show that the purity of the target protein in the elution buffer after preliminary separation is >50%. The protein recovery rate after one-step affinity chromatography is calculated to be 8.1%, and the protein loading capacity of the one-step affinity chromatography is 76.55 mg / mL column material.

[0112] Example 4: Anion Exchange Chromatography of Bst DNA Polymerase

[0113] Take 25g of anion chromatography medium Q Sepharose FF and load it into XK 16 / 20 chromatography column. The following chromatography processes are all performed on AKTA protein purifier. First, use 2-3CV 2M NaCl, 0.5M NaOH to wash the anion chromatography medium, then use 5-10CV deionized water to wash the anion chromatography medium until the pH is neutral, and then use 5-10CV equilibration buffer (20mMTris-HCl, 100mM KCl, 5% glycerol and 0.1% Tween 20 pH 7.5) to equilibrate the chromatography column. The eluate obtained in Example 3 was loaded onto the above-mentioned balanced chromatography column. After loading, 5CV of equilibration buffer was used to wash the impurities that were not attached to the column. 5-10CV of impurity wash buffer (20mM Tris-HCl, 200mM KCl, 5% glycerol and 0.1% Tween 20, pH 7.5) was used to wash away the impurities that were weakly bound to the column material. 5-10CV of elution buffer (20mM Tris-HCl, 250mM KCl, 5% glycerol and 0.1% Tween 20, pH 7.5) was used to elute the target protein and collect it. 3-5CV of 2M NaCl was used to wash the proteins that were strongly bound to the column material. 3-5CV of 0.5M NaOH was used to wash the column material. Finally, deionized water was used to wash the column material until the pH was neutral. Finally, the column material was stored in 20% ethanol. The collected eluate containing the target protein was subjected to SDS-PAGE gel electrophoresis. The detection results are shown in FIG. Figure 2 As shown. Figure 2 It can be seen that some of the host's own impurities have been removed after the two-step anion chromatography. The test results show that the purity of the target protein in the elution buffer after preliminary separation is >70%. The calculated protein recovery rate of the two-step chromatography is 56.34%, and the two-step protein loading capacity is 5.07 mg / mL column material.

[0114] Example 5: Hydrophobic chromatography of Bst DNA polymerase

[0115] Take 25g of hydrophobic chromatography medium Phenyl Sepharose High Performance and load it on XK16 / 20, use 2-3CV 0.5M NaOH to pretreat the chromatography filler, wash with deionized water to neutral pH as described above, and then use 5-10CV equilibration buffer (20mM Tris-HCl, 0.85M (NH4)2SO4, 250mM KCl and 5% glycerol, pH 7.5) to equilibrate the chromatography column, add (NH4)2SO4 to the two-step elution component, i.e., the elution component collected in Example 4, to a final concentration of 0.85M, and a small amount of precipitate is generated after adding (NH4)2SO4, which is filtered through a 0.45μm filter membrane and then loaded onto the equilibrated chromatography column. After loading, use 5CV equilibration buffer to wash away the residual impurities on the column material, and use 5-10CV elution buffer (20mM Tris-HCl, 0.6M (NH4)2SO4 and 5% glycerol pH 7.5) Elute the target protein and collect it, then use 3-5CV deionized water and 3-5CV 0.5M NaOH to wash the chromatography filler, and store it in 20% ethanol. Perform SDS-PAGE gel electrophoresis on the collected eluate containing the target protein, and the detection results are as follows: Figure 3 As shown. Combined Figure 3 The protein recovery rate of the three-step hydrophobic chromatography was calculated to be 23.61%, and the protein loading capacity of the three-step hydrophobic chromatography was 4.96 mg / mL column material. The test results showed that the purity of the target protein in the elution buffer after purification was >95%.

[0116] Example 6: Post-treatment of Bst DNA polymerase

[0117] According to the protein content in the three-step elution fraction, 0.1 m 2 For the 10kDa ultrafiltration membrane package, first use 0.5MNaOH to circulate and flush the ultrafiltration membrane package for more than 30 minutes to disinfect and remove pyrogens, then rinse the membrane package with ultrapure water until the pH is neutral, then use displacement buffer (20mM Tris-HCl, 100mM KCl, pH 7.5) to rinse the membrane package until pH 7.5±0.5 is filtered out. After the eluted components collected in Example 6 are concentrated to the dead volume of the membrane package, a displacement buffer of 2 times the dead volume of the membrane package is added for displacement. The number of displacements is 3 to 5 times, and the displacement end point is pH 7.5±0.5. The deviation between the conductivity and the displacement buffer is no more than 1mS / cm. Accurately measure a certain volume of the ultrafiltration displacement semi-finished product, add glycerol in a 1:1 volume ratio, mix thoroughly, and filter using a 0.22μm sterile filter to obtain the finished product. Please refer to Figure 4 The finished product was subjected to SDS-PAGE gel electrophoresis and the quality of the finished product obtained by the above process was analyzed. Figure 5The test results are shown in Table 1. As can be seen from Table 1, the Bst DNA polymerase prepared by the process of Example 6 has no DNA residue, no RNase and Nickase pollution. Table 1 is the test results of host DNA residues of the finished product obtained in Example 7, where ERC represents the spiked sample.

[0118] Table 1 Detection results of host DNA residues in the finished products obtained in Example 6

[0119]

[0120] Example 7: Activity and stability detection of Bst DNA polymerase

[0121] One unit of Bst DNA polymerase activity is the amount of enzyme required to incorporate 25 nmol of dNTP into acid-insoluble substances within 30 min at 65°C. The activity of Bst DNA polymerase and samples after stability treatment were measured using the Qubit ssDNA assay kit;

[0122] Thermal stability treatment method: Take 50-200 μL of the finished Bst DNA polymerase and incubate it in a 1 mL EP tube without a heat source at 37°C for 7 days to test the residual activity;

[0123] Freeze-thaw stability treatment method: Take 50-200μL Bst DNA polymerase finished product, place it in a 1mL EP tube without heat source, place it in a -80℃ refrigerator and freeze it repeatedly for 10 times to test the residual activity; please refer to Figure 6 .

[0124] The above-mentioned process for removing HCD provided in the present application, after nuclease enzymolysis and screening and combination of various chromatographic fillers, obtains the purification process conditions of pre-treatment (i.e., obtaining crude enzyme solution and enzymatic solution), three-step chromatography and post-treatment (i.e., ultrafiltration replacement and preservation), the pre-treatment can solve the problem of difficulty in filtering after bacterial mud crushing and more HCD and HCP in crude enzyme solution, and this step is easy to scale production; the three-step chromatography process is affinity chromatography, anion exchange chromatography and hydrophobic chromatography, the process connection is easy, and it is easy to linearly amplify; and after post-treatment, Bst DNA polymerase without nucleic acid residue can be output. The above preparation method successfully prepared Bst DNA polymerase with no host DNA residue and enzyme purity up to 95%, high purity, no RNase and Nickase contamination. The preparation method of the above Bst DNA polymerase has the advantages of simple operation method, high yield, high protein purity, high enzyme activity, and easy to amplify production. The results of property evaluation compared with mainstream products on the market show that the Bst DNA polymerase purified by the present invention has better reproducibility, thermal stability and freeze-thaw stability.

[0125] The above are only preferred embodiments of the present application, and only specifically describe the technical principles of the present application. These descriptions are only for explaining the principles of the present application and cannot be interpreted as limiting the scope of protection of the present application in any way. Based on the explanation here, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application, and other specific implementation methods of the present application that can be associated with the technicians in this field without creative work, should be included in the scope of protection of the present application.

Claims

1. A chromatography process for removing host nucleic acid residues in Bst DNA polymerase, characterized in that: The steps include: Performing precipitation treatment on the crude enzyme solution to obtain a crude enzyme supernatant; treating the crude enzyme supernatant with nuclease to obtain an enzymatic hydrolyzate; performing affinity chromatography on the enzymatic hydrolysate to obtain a first eluted sample; performing anion exchange chromatography on the first eluted sample to obtain a second eluted sample; performing hydrophobic chromatography on the second eluted sample to obtain a third eluted sample; The third eluted sample is concentrated and ultrafiltered to obtain a pure Bst DNA polymerase product without residual host nucleic acid.

2. The chromatography process for removing host nucleic acid residues in Bst DNA polymerase according to claim 1, characterized in that: Before the step of precipitating the crude enzyme solution to obtain the crude enzyme supernatant, the method also includes the step of preparing the crude enzyme solution, which specifically includes the following steps: crushing the fermented bacterial sludge of the Bst DNA polymerase, adding a flocculant or a water-soluble cationic polymer with a final volume concentration of 0.1%-1%, standing for solid-liquid separation, collecting the supernatant, and obtaining the crude enzyme supernatant.

3. The chromatography process for removing host nucleic acid residues in Bst DNA polymerase according to claim 1, characterized in that: The step of subjecting the crude enzyme supernatant to nuclease treatment to obtain an enzymatic solution specifically includes the following steps: adjusting the pH of the crude enzyme supernatant to 8.0-9.0, adding 1-5 mM MgCl2 and 200 U / g bacteria-1000 U / g bacteria nuclease to the supernatant, and reacting at 37°C for 1 hour.

4. The chromatography process for removing host nucleic acid residues in Bst DNA polymerase according to claim 1, characterized in that: The step of performing affinity chromatography on the enzymatic hydrolysate to obtain a first eluted sample specifically includes the following steps: Equilibrium affinity chromatography column; After adding 300mM-500mM NaCl to the enzymatic hydrolysate, the sample is loaded onto the equilibrated affinity chromatography column; Carrying out a washing treatment on the affinity chromatography column after loading the sample; The affinity chromatography column after the washing treatment is eluted with a first elution buffer to obtain the first eluted sample.

5. The chromatography process for removing host nucleic acid residues in Bst DNA polymerase according to claim 4, characterized in that: The affinity chromatography column is a Ni column affinity chromatography, and the affinity chromatography medium is selected from any one of Ni Sepharose HP, Ni Sepha rose6FF, TALON Superflow, and Ni Sepharose excel; The pH of the equilibration buffer used to balance the affinity chromatography column is 7.0-8.0, and includes 10mM-50mM TrisHCl, 300mM-500mM NaCl, and 10mM-50mM imidazole; The pH of the washing buffer used for washing the affinity chromatography column after loading is 7.0-8.0, and includes 10mM-50mM Tris HCl and 500mM-2M NaCl; The first elution buffer has a pH of 7.0-8.0 and includes 10 mM-50 mM Tris HCl, 50 mM-200 mM NaCl, and 100 mM-200 mM imidazole.

6. The chromatography process for removing host nucleic acid residues in Bst DNA polymerase according to claim 1, characterized in that: The step of subjecting the first eluted sample to anion exchange chromatography to obtain a second eluted sample specifically includes the following steps: Equilibrating anion exchange chromatography columns; directly loading the first eluted sample onto the equilibrated anion exchange chromatography column; The loaded anion exchange chromatography column is eluted with a second elution buffer to obtain the second eluted sample.

7. The chromatography process for removing host nucleic acid residues in Bst DNA polymerase according to claim 6, characterized in that: The chromatography medium used is selected from any one of Q Sepharose FF, Q Sepharose 4FF, Q Sepharose XL, Q Sepharose HP, Q Sepharose Big Beads, Capto Q, DEAE Sepharose FF, DEAE Sepharose CL 6B, DEAESephacel, Capto DEAE, and Anx Sepharose 4FF; The second elution buffer has a pH of 7.0 to 8.0 and includes 10 mM to 50 mM Tris HCl and 150 mM to 250 mM NaCl; The pH of the balancing buffer for balancing the anion exchange chromatography column is 7.0-8.0, and includes 10mM-50mM TrisHCl and 50mM-250mM NaCl.

8. The chromatography process for removing host nucleic acid residues in Bst DNA polymerase according to claim 1, characterized in that: The step of performing hydrophobic chromatography on the second eluted sample to obtain a third eluted sample specifically includes the following steps: Equilibrating a hydrophobic chromatography column; After adding 0.8M to 1.2M ammonium sulfate solid to the first elution buffer to make the final concentration of 0.8-1.2M ammonium sulfate, the sample is loaded onto the equilibrated hydrophobic chromatography column; The loaded hydrophobic chromatography column is eluted with a third elution buffer to obtain the third elution sample.

9. The chromatography process for removing host nucleic acid residues in Bst DNA polymerase according to claim 8, characterized in that: The chromatography medium used is selected from any one of Capto Phenyl FF, Phenyl Sepharose 6Fast Flow, Capto PhenylImpRes, and Phenyl Sepharose High Performance; the pH of the third elution buffer is 7.0-8.0, and includes 10mM-50mM Tris HCl and 0.1M-0.5M ammonium sulfate; the pH of the balancing buffer for balancing the hydrophobic ion exchange chromatography column is 7.0-8.0, and includes 10mM-50mM Tris HCl and 0.8M-1M ammonium sulfate.

10. A Bst DNA polymerase, characterized in that The method is prepared by the chromatography process for removing host nucleic acid residues in Bst DNA polymerase according to any one of claims 1 to 9.

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