Protein purification partner as well as application and use method thereof

Abstract

The invention provides a protein purification partner as well as application and a use method thereof. The protein purification partner comprises all-round nuclease and a precipitant, and the precipitant is a cationic flocculant; the protein purification partner disclosed by the invention can be used for reducing the viscosity of feed liquid caused by nucleic acid, shortening the treatment time of resuspension and filtration and increasing the protein yield; the method can be used for removing exogenous nucleic acid of recombinant protein products, reducing the risk of residual toxicity of nucleic acid and improving the safety of the products; the method can be used for removing nucleic acid wound on the surfaces of particles (viruses, inclusion bodies and the like), and release and purification of the particles are facilitated; the method can be used for removing part of impure proteins and improving the purity and specific activity of target proteins; the clarity of the sample can be improved, and the problem of difficult filtration is effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical field of protein purification, and in particular to a protein purification partner and an application and use method thereof. Background Art

[0002] In the purification process of soluble recombinant proteins expressed in E. coli, yeast and other cells, there are often problems such as difficulty in resuspending the liquid, long time consumption, and high host nucleic acid residues. The above phenomenon can be improved by adding precipitants in the pre-purification treatment stage.

[0003] As disclosed in the prior art, a composition and method for high-efficiency protein precipitation is prepared by adding a non-volatile precipitant to a protein aqueous solution at a low concentration. Water is then removed from the resulting solution until the precipitant and protein content in the solution increases to a concentration that provides the desired separation of the protein between the supernatant and the precipitate, and additional water can be removed from the supernatant to provide additional fractional separation; wherein the non-volatile precipitant includes an organic acid, an organic acid salt (e.g., sodium citrate), an inorganic salt, and a hydrophilic polymer. However, only a single precipitant is used in this solution, and the improvement effect is limited.

[0004] The prior art also discloses a method for efficiently treating a clarification and harvesting cell fluid with a high solid content. Specifically, when treating a cell fluid with a high solid content, the cell fluid is first centrifuged using a centrifuge and then filtered using a membrane package. Alternatively, a flocculant is added after centrifugation for flocculation and then filtered using a membrane package. This method can be used to clarify and harvest cell culture fluid with a solid content of 15% or more. The flocculant used includes a strong cationic polyelectrolyte (polydiallyldimethylammonium chloride or butyl polyethylene bromide). Similarly, only a single flocculant is used in this solution, and the improvement effect is limited.

[0005] In summary, the existing technologies all use a single precipitant or flocculant for treatment in the pre-purification treatment stage, but the improvement effect on the problems of difficult and time-consuming resuspension process of the feed liquid and high host nucleic acid residue is limited. Based on this, it is necessary to make improvements. Summary of the invention

[0006] The present invention provides a protein purification partner and its application and use method, so as to solve or at least partially solve the defects in the prior art.

[0007] In a first aspect, the present invention provides a protein purification chaperone, comprising: a universal nuclease and a precipitant, wherein the precipitant is a cationic flocculant.

[0008] Preferably, the cationic flocculant includes at least one of polyacrylamide, polyaluminum ferric silicate, polyaluminum ferric chloride, and polyaluminum phosphate.

[0009] In a second aspect, the present invention also provides an application of the protein purification partner in the purification of soluble recombinant proteins expressed in bacterial cells.

[0010] In a third aspect, the present invention further provides a method for using the protein purification partner, comprising the following steps:

[0011] Obtaining bacterial cells;

[0012] Adding bacterial cells to the resuspension solution to obtain a bacterial resuspension solution;

[0013] Add the universal nuclease to the bacterial resuspension, centrifuge, and collect the precipitate;

[0014] Add cell lysis solution to the precipitate for lysis, and then add the precipitant.

[0015] Preferably, the pH of the bacterial resuspension is adjusted to 6.0-11.0, and the temperature is controlled to 0-45°C, and then the universal nuclease is added.

[0016] Preferably, a cell lysis solution is added to the precipitate for lysis. After the lysis is completed, the pH is adjusted to 6.0-11.0, and the temperature is controlled to 2-30° C., and then a precipitant is added.

[0017] Preferably, the omnipotent nuclease described in claim 1 is added to the bacterial resuspension, treated for 0.5 to 2 hours, centrifuged, and the precipitate is collected; wherein the amount of the omnipotent nuclease added is: 20 to 3000 U of omnipotent nuclease per gram of bacterial cells;

[0018] And / or, adding cell lysis solution to the precipitate for lysis, the lysis time is 1 to 12 hours, and then adding the precipitant; wherein the precipitant is added in the form of a precipitant aqueous solution, the mass concentration of the precipitant aqueous solution is 0.1 to 1%, and 0.02 to 0.5 mL of the precipitant aqueous solution is added per gram of bacterial cells.

[0019] Preferably, 500 U of universal nuclease is added per gram of bacterial cells, and 0.15 to 0.2 mL of precipitant aqueous solution is added per gram of bacterial cells.

[0020] Preferably, the precipitant aqueous solution comprises a polyacrylamide aqueous solution with a mass concentration of 0.1-1% and a polyaluminum ferric silicate aqueous solution with a mass concentration of 0.1-1%, and the volume ratio of the polyacrylamide aqueous solution to the polyaluminum ferric silicate aqueous solution is 1:(2-4).

[0021] Preferably, in the step of adding the bacterial cells to the resuspension, the resuspension is a PBS buffer or a Tris-HCl buffer, the pH of the resuspension is 7.0-8.0; the mass volume ratio of the bacterial cells to the resuspension is 1 g: (10-20) mL;

[0022] And / or, the cell lysis solution includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water; the mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is (0.04-0.05) g: (0.025-0.035) g: (0.04-0.05) g: (0.02-0.03) g: (1.5-2.0) mL: (200-250) mL; the concentration of Tris-HCl is 18-22 mM;

[0023] and / or, the mass volume ratio of the precipitate to the cell lysate is (1-2) g:(5-10) mL;

[0024] And / or, the bacterial cells include Escherichia coli cells or yeast cells.

[0025] The protein purification companion and its application and use method of the present invention have the following beneficial effects compared with the prior art:

[0026] The protein purification partner of the present invention comprises an all-purpose nuclease and a precipitant, wherein the precipitant is a cationic flocculant; the protein purification partner of the present invention can be used to reduce the viscosity of the feed solution caused by nucleic acids, shorten the processing time of resuspension and filtration, and increase the protein yield; can be used to remove exogenous nucleic acids in recombinant protein products, reduce the risk of residual nucleic acid toxicity, and improve product safety; can be used to remove nucleic acids entangled on the surface of particles (viruses, inclusion bodies, etc.), which is beneficial to the release and purification of particles; can be used to remove some impurities and improve the purity and specific activity of target proteins; can improve the clarity of samples and effectively solve the problem of difficult filtration. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 This is a state diagram after Escherichia coli cells are resuspended according to the methods in Example 1 and Comparative Example 1;

[0029] Figure 2 According to the method in Examples 2 to 4 and Comparative Examples 2 to 4, lysis is performed, and the supernatant is taken after lysis, and the state diagram of the supernatant;

[0030] Figure 3 The electrophoretic diagram of the supernatant after lysis according to the method in Comparative Example 4 and Comparative Examples 6 to 11;

[0031] Figure 4 The electrophoretic diagrams of the supernatant after lysis according to the methods in Example 2, Examples 5-6, Comparative Example 5 and Comparative Example 2 are shown. DETAILED DESCRIPTION

[0032] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0034] In the description of the present invention, it should be understood that the directions or positions indicated by “upper” and the like are based on the directions or positions shown in the accompanying drawings, or are the directions or positions in which the product of the invention is usually placed when in use, or are directions or positions commonly understood by those skilled in the art. These directions or positions are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or component referred to must have a specific direction, be constructed and operated in a specific direction, and therefore should not be understood as a limitation on the present invention.

[0035] The following are described in detail. It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of the present application, the term "including" means "including but not limited to". Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and simplicity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the numbered ranges, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated in this article, it is meant to include any quoted numbers (fractions or integers) within the indicated range.

[0036] The invention provides a protein purification partner, comprising: a universal nuclease and a precipitant, wherein the precipitant is a cationic flocculant.

[0037] The protein purification partner of the present invention is a precipitant which is a cationic flocculant, which can quickly combine with nucleic acid substances to precipitate them, reduce the viscosity of the feed solution and the amount of nucleic acid residues, and improve the purity of the target protein; the all-purpose nuclease can degrade double-stranded, single-stranded, linear, and circular DNA and RNA under a very wide range of conditions, thereby significantly reducing the viscosity of the bacterial solution; the present invention can greatly improve the problems of difficult and time-consuming feed solution resuspension process, high host nucleic acid residues, etc. in the purification process of soluble recombinant proteins expressed in Escherichia coli, yeast, etc. by combining the all-purpose nuclease and the precipitant.

[0038] Specifically, the universal nuclease of the present invention is a commercially available universal nuclease, for example, the universal nuclease of Hanhai New Enzyme Biotechnology Co., Ltd., item number: HBP000109, CAS: 9025-65-4, specification 5KU.

[0039] In some embodiments, the cationic flocculant includes at least one of polyacrylamide, polyaluminum ferric silicate, polyaluminum ferric chloride, and polyaluminum phosphate.

[0040] Based on the same inventive concept, the present invention also provides an application of the above-mentioned protein purification partner in the purification of soluble recombinant proteins expressed in bacterial cells.

[0041] The protein purification partner of the present invention can be used to reduce the viscosity of the feed solution caused by nucleic acids, shorten the processing time of resuspension and filtration, and increase the protein yield; can be used to remove exogenous nucleic acids in recombinant protein products, reduce the risk of residual nucleic acid toxicity, and improve product safety; can be used to remove nucleic acids entangled on the surface of particles (viruses, inclusion bodies, etc.), which is beneficial to the release and purification of particles; can be used to remove some impurities and improve the purity and specific activity of target proteins; can improve the clarity of samples and effectively solve the problem of difficult filtration.

[0042] Based on the same inventive concept, the present invention also provides a method for using the above-mentioned protein purification partner, comprising the following steps:

[0043] S1. Obtain bacterial cells;

[0044] S2, adding bacterial cells to the resuspension solution to obtain a bacterial resuspension solution;

[0045] S3, adding the above-mentioned universal nuclease to the bacterial resuspension, centrifuging, and collecting the precipitate;

[0046] S4. Add cell lysis buffer to the precipitate for lysis, and then add the above-mentioned precipitant.

[0047] The method for using the protein purification companion of the present invention comprises the following steps: adding a universal nuclease during the bacterial cell resuspension stage, which can remove nucleic acids entangled on the surface of particles (viruses, inclusion bodies, etc.), facilitate the release and purification of the particles, and at the same time reduce the viscosity of the feed liquid caused by the nucleic acid to accelerate the bacterial sludge resuspension process, has the effect of significantly hydrolyzing and precipitating nucleic acids, and can largely remove host nucleic acid residues; adding a cell lysis solution to the precipitate for lysis, and then adding a precipitant, which can greatly improve the clarity of the feed liquid and improve the problem of difficult filtration.

[0048] In some embodiments, the pH of the bacterial resuspension is adjusted to 6.0-11.0, and the temperature is controlled at 0-45°C, and then the universal nuclease is added. Preferably, the pH of the bacterial resuspension is adjusted to 8.0-9.2, and the temperature is controlled at 37°C, and then the universal nuclease is added.

[0049] In some embodiments, a cell lysis solution is added to the precipitate for lysis. After the lysis is completed, the pH is adjusted to 6.0-11.0, and the temperature is controlled to 2-30°C, and then a precipitant is added. Preferably, a cell lysis solution is added to the precipitate for lysis. After the lysis is completed, the pH is adjusted to 8.0-9.2, and the temperature is controlled to 25°C, and then a precipitant is added.

[0050] In some embodiments, the above-mentioned universal nuclease is added to the bacterial resuspension, treated for 0.5 to 2 hours, centrifuged, and the precipitate is collected; wherein the amount of the universal nuclease added is: 20 to 3000 U of universal nuclease per gram of bacterial cells.

[0051] In some embodiments, a cell lysis solution is added to the precipitate for lysis, the lysis time is 1 to 12 hours, and then a precipitant is added; wherein the precipitant is added in the form of an aqueous precipitant solution, the mass concentration of the aqueous precipitant solution is 0.1 to 1%, and 0.02 to 0.5 mL of the aqueous precipitant solution is added per gram of bacterial cells.

[0052] In some embodiments, 500 U of universal nuclease is added per gram of bacterial cells, and 0.15-0.2 mL of precipitant aqueous solution is added per gram of bacterial cells.

[0053] Preferably, the precipitant includes a polyacrylamide aqueous solution with a mass concentration of 0.1 to 1% and a polyaluminum ferric silicate aqueous solution with a mass concentration of 0.1 to 1%, and the volume ratio of the polyacrylamide aqueous solution to the polyaluminum ferric silicate aqueous solution is 1:(2 to 4). More preferably, the volume ratio of the polyacrylamide aqueous solution to the polyaluminum ferric silicate aqueous solution is 1:3.

[0054] In some embodiments, in the step of adding bacterial cells to a resuspension, the resuspension is a PBS buffer or a Tris-HCl buffer, and the pH of the resuspension is 7.0-8.0; the mass volume ratio of the bacterial cells to the resuspension is 1 g: (10-20) mL, and preferably, the resuspension is a 20 mM Tris-HCl buffer with a pH of 7.5.

[0055] In some embodiments, the cell lysis solution includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water; wherein the mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is (0.04-0.05) g:(0.025-0.035) g:(0.04-0.05) g:(0.02-0.03) g:(1.5-2.0) mL:(200-250) mL; the concentration of Tris-HCl is 18-22 mM.

[0056] In some embodiments, the mass volume ratio of the precipitate to the cell lysate is (1-2) g: (5-10) mL;

[0057] In some embodiments, the bacterial cells include Escherichia coli cells or yeast cells.

[0058] The following further illustrates the protein purification partner of the present application and its application and use method with specific examples. This section further illustrates the content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0059] In the following examples, the universal nuclease is the universal nuclease of Hanhai New Enzyme Biotechnology Co., Ltd., item number; HBP000109, specification 5KU; the cationic flocculant polyacrylamide was purchased from Shandong Kunxin Environmental Protection Technology Co., Ltd., with a molecular weight of 8-12 million; the cationic flocculant polyaluminum chloride ferric was purchased from Henan Hongchang Chemical Co., Ltd.; the cationic flocculant polyaluminum silicate ferric was purchased from Gongyi Jiaxin Water Purification Materials Co., Ltd.

[0060] Example 1

[0061] The embodiment of the present application provides a protein purification partner, including: a universal nuclease and a precipitant, the precipitant is a cationic flocculant, and the cationic flocculant is polyacrylamide.

[0062] This embodiment also provides a method for using the above protein purification partner, comprising the following steps:

[0063] S1. Obtain Escherichia coli cells;

[0064] S2, adding Escherichia coli cells to the resuspension to obtain a resuspension of cells; the resuspension is a 20 mM Tris-HCl buffer with a pH of 7.5, and the mass volume ratio of the Escherichia coli cells to the resuspension is 1 g:15 mL;

[0065] S3, adjusting the pH of the bacterial resuspension to 8.5, and controlling the temperature to 37°C, then adding universal nuclease for treatment for 1 hour, centrifuging, and collecting the precipitate; wherein the amount of universal nuclease added is: adding 800U universal nuclease per gram of Escherichia coli;

[0066] S4. Add cell lysis buffer to the precipitate for lysis, lyse for 1.5 hours, adjust the pH to 8.5, and control the temperature to 25°C, then add a polyacrylamide aqueous solution with a mass concentration of 0.1%, and add 0.05 mL of polyacrylamide aqueous solution for every gram of Escherichia coli cells; the cell lysis buffer includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water; wherein the mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is 0.04 g: 0.03 g: 0.04 g: 0.02 g: 2 mL: 250 mL; the concentration of Tris-HCl is 20 mM; the mass volume ratio of the precipitate to the cell lysis buffer is 1 g: 10 mL.

[0067] Comparative Example 1

[0068] This comparative example provides a method for resuspending Escherichia coli, comprising the following steps:

[0069] S1. Obtain Escherichia coli cells;

[0070] S2. Add the Escherichia coli cells to the resuspension to obtain a resuspension of the cells; the resuspension is a 20 mM Tris-HCl buffer with a pH of 7.5, and the mass volume ratio of the Escherichia coli cells to the resuspension is 1 g:15 mL.

[0071] The state of Escherichia coli cells after resuspension according to the methods in Example 1 and Comparative Example 1 is as follows: Figure 1 As shown, Figure 1 The control group and experimental group in a and b respectively represent Example 1 and Comparative Example 1. The control group in a represents the state in which the Escherichia coli cells are resuspended after stirring according to the method in Comparative Example 1 (without adding the omnipotent nuclease), and the experimental group in a represents the state in which the Escherichia coli cells are resuspended after stirring 1 hour after adding the omnipotent nuclease according to the method in Example 1; the control group in b represents the state in which the Escherichia coli cells are resuspended after standing according to the method in Comparative Example 1 (without adding the omnipotent nuclease), and the experimental group in b represents the state in which the Escherichia coli cells are resuspended after stirring 1 hour after adding the omnipotent nuclease according to the method in Example 1.

[0072] from Figure 1 It can be clearly seen that the resuspension state of the experimental group is very uniform, while the control group has obvious clumping bacteria, which shows that the addition of omnipotent nuclease can improve the resuspension state of E. coli.

[0073] Example 2

[0074] The embodiment of the present application provides a protein purification partner, including: a universal nuclease and a precipitant, the precipitant is a cationic flocculant, and the cationic flocculant includes polysilicate aluminum iron and polyacrylamide.

[0075] This embodiment also provides a method for using the above protein purification partner, comprising the following steps:

[0076] S1. Obtain Escherichia coli cells;

[0077] S2, adding Escherichia coli cells to the resuspension to obtain a resuspension of cells; the resuspension is a 20 mM Tris-HCl buffer with a pH of 7.5, and the mass volume ratio of the Escherichia coli cells to the resuspension is 1 g:15 mL;

[0078] S3, adjusting the pH of the bacterial resuspension to 8.5, and controlling the temperature to 37°C, then adding universal nuclease for treatment for 1 hour, centrifuging, and collecting the precipitate; wherein, the amount of universal nuclease added is: adding 500U universal nuclease per gram of Escherichia coli;

[0079] S4. Add cell lysis buffer to the precipitate for lysis, lyse for 1.5 hours, adjust the pH to 8.5, and control the temperature to 25°C, then add a polyacrylamide aqueous solution with a mass concentration of 0.1% and a polyaluminum ferric silicate aqueous solution with a mass concentration of 0.1%, and add 0.05mL of polyacrylamide aqueous solution and 0.15mL of polyaluminum ferric silicate aqueous solution for every gram of Escherichia coli cells; the cell lysis buffer includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water; wherein the mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is 0.04g:0.03g:0.04g:0.02g:2mL:250mL; the concentration of Tris-HCl is 20mM; the mass volume ratio of the precipitate to the cell lysis buffer is 1g:10mL.

[0080] Comparative Example 2

[0081] This comparative example provides a method for treating Escherichia coli cells, comprising the following steps:

[0082] S1. Obtain Escherichia coli cells;

[0083] S2, adding Escherichia coli cells to the resuspension to obtain a resuspension of cells; the resuspension is a 20 mM Tris-HCl buffer with a pH of 7.5, and the mass volume ratio of the Escherichia coli cells to the resuspension is 1 g:15 mL;

[0084] S3, centrifuge the bacterial resuspension and collect the precipitate;

[0085] S4. Add cell lysis buffer to the precipitate for lysis for 1.5 hours. The cell lysis buffer includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water. The mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is 0.04g:0.03g:0.04g:0.02g:2mL:250mL. The concentration of Tris-HCl is 20mM. The mass volume ratio of precipitate to cell lysis buffer is 1g:10mL.

[0086] Example 3

[0087] The embodiment of the present application provides a protein purification partner, including: a universal nuclease and a precipitant, the precipitant is a cationic flocculant, and the cationic flocculant is polysilicate aluminum iron.

[0088] This embodiment also provides a method for using the above protein purification partner, comprising the following steps:

[0089] S1. Obtain Escherichia coli cells;

[0090] S2, adding Escherichia coli cells to the resuspension to obtain a resuspension of cells; the resuspension is a 20 mM Tris-HCl buffer with a pH of 7.5, and the mass volume ratio of the Escherichia coli cells to the resuspension is 1 g:15 mL;

[0091] S3, adjusting the pH of the bacterial resuspension to 8.5, and controlling the temperature to 37°C, then adding universal nuclease for treatment for 1 hour, centrifuging, and collecting the precipitate; wherein, the amount of universal nuclease added is: adding 500U universal nuclease per gram of Escherichia coli;

[0092] S4. Add cell lysis solution to the precipitate for lysis for 1.5 hours, adjust the pH to 8.5, and control the temperature to 25°C, then add a 0.1% polysilicate aluminum ferric aqueous solution, and add 0.2 mL of polysilicate aluminum ferric aqueous solution per gram of Escherichia coli cells; the cell lysis solution includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water; wherein the mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is 0.04g:0.03g:0.04 g:0.02g:2mL:250mL; the concentration of Tris-HCl is 20mM; the cell lysis solution includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water; wherein the mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is 0.04g:0.03g:0.04g:0.02g:2mL:250mL; the concentration of Tris-HCl is 20mM; the mass volume ratio of the precipitate to the cell lysis solution is 1g:10mL.

[0093] Comparative Example 3

[0094] This comparative example provides a method for treating Escherichia coli cells, comprising the following steps:

[0095] S1. Obtain Escherichia coli cells;

[0096] S2, adding Escherichia coli cells to the resuspension to obtain a resuspension of cells; the resuspension is a 20 mM Tris-HCl buffer with a pH of 7.5, and the mass volume ratio of the Escherichia coli cells to the resuspension is 1 g:10 mL;

[0097] S3, centrifuge the bacterial resuspension and collect the precipitate;

[0098] S4. Add cell lysis buffer to the precipitate for lysis for 1.5 hours. The cell lysis buffer includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water. The mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is 0.04g:0.03g:0.04g:0.02g:2mL:250mL. The concentration of Tris-HCl is 20mM. The mass volume ratio of precipitate to cell lysis buffer is 1g:8mL.

[0099] Example 4

[0100] The embodiment of the present application provides a protein purification partner, including: a universal nuclease and a precipitant, the precipitant is a cationic flocculant, and the cationic flocculant is polyacrylamide.

[0101] This embodiment also provides a method for using the above protein purification partner, comprising the following steps:

[0102] S1. Obtain Escherichia coli cells;

[0103] S2, adding Escherichia coli cells to the resuspension to obtain a resuspension of cells; the resuspension is a 20 mM Tris-HCl buffer with a pH of 7.5, and the mass volume ratio of the Escherichia coli cells to the resuspension is 1 g:15 mL;

[0104] S3, adjusting the pH of the bacterial resuspension to 8.5, and controlling the temperature to 37°C, then adding universal nuclease for treatment for 1 hour, centrifuging, and collecting the precipitate; wherein, the amount of universal nuclease added is: adding 500U universal nuclease per gram of Escherichia coli;

[0105] S4. Add cell lysis buffer to the precipitate for lysis, lyse for 1.5 hours, adjust the pH to 8.5, and control the temperature to 25°C, then add a polyacrylamide aqueous solution with a mass concentration of 0.1%, and add 0.2 mL of polyacrylamide aqueous solution for every gram of Escherichia coli cells; the cell lysis buffer includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water; wherein the mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is 0.04 g: 0.03 g: 0.04 g: 0.02 g: 2 mL: 250 mL; the concentration of Tris-HCl is 20 mM, and the mass volume ratio of the precipitate to the cell lysis buffer is 1 g: 10 mL.

[0106] Comparative Example 4

[0107] This comparative example provides a method for treating Escherichia coli cells, comprising the following steps:

[0108] S1. Obtain Escherichia coli cells;

[0109] S2, adding Escherichia coli cells to the resuspension to obtain a resuspension of cells; the resuspension is a 20 mM Tris-HCl buffer with a pH of 7.5, and the mass volume ratio of the Escherichia coli cells to the resuspension is 1 g:10 mL;

[0110] S3, centrifuge the bacterial resuspension and collect the precipitate;

[0111] S4. Add cell lysis buffer to the precipitate for lysis for 1.5 hours. The cell lysis buffer includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water. The mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is 0.04g:0.03g:0.04g:0.02g:2mL:250mL. The concentration of Tris-HCl is 20mM. The mass volume ratio of precipitate to cell lysis buffer is 1g:5mL.

[0112] The cells were lysed according to the methods in Examples 2 to 4 and Comparative Examples 2 to 4. After lysing, the supernatant was taken and the state of the supernatant was observed. The results were as follows: Figure 2 shown.

[0113] Figure 2 Among them, the item A does not contain the purified partner corresponding to the proportion 2, and contains the purified partner corresponding to Example 2; the item B does not contain the purified partner corresponding to the proportion 3, and contains the purified partner corresponding to Example 3; the item C does not contain the purified partner corresponding to the proportion 4, and contains the purified partner corresponding to Example 4.

[0114] from Figure 2 It can be seen that according to the methods in Examples 2 to 4, the clarity of the supernatant after lysis is effectively improved; wherein, according to the method in Example 2, the clarity of the supernatant is improved by 94.8%; according to the method in Example 3, the clarity of the supernatant is improved by 91.7%; according to the method in Example 4, the clarity of the supernatant is improved by 88.7%.

[0115] Example 5

[0116] The embodiment of the present application provides a protein purification partner, including: a universal nuclease and a precipitant, the precipitant is a cationic flocculant, and the cationic flocculant includes polysilicate aluminum iron and polyacrylamide.

[0117] This embodiment also provides a method for using the above protein purification partner, comprising the following steps:

[0118] S1. Obtain Escherichia coli cells;

[0119] S2, adding Escherichia coli cells to the resuspension to obtain a resuspension of cells; the resuspension is a 20 mM Tris-HCl buffer with a pH of 7.5, and the mass volume ratio of the Escherichia coli cells to the resuspension is 1 g:15 mL;

[0120] S3, adjusting the pH of the bacterial resuspension to 8.5, and controlling the temperature to 37°C, then adding universal nuclease for treatment for 1 hour, centrifuging, and collecting the precipitate; wherein, the amount of universal nuclease added is: adding 500U universal nuclease per gram of Escherichia coli;

[0121] S4. Add cell lysis buffer to the precipitate for lysis, lyse for 1.5 hours, adjust the pH to 8.5, and control the temperature to 25°C, then add a polyacrylamide aqueous solution with a mass concentration of 0.1% and a polyaluminum-iron silicate aqueous solution with a mass concentration of 0.1%, and add 0.025mL of polyacrylamide aqueous solution and 0.075mL of polyaluminum-iron silicate aqueous solution for every gram of Escherichia coli cells; the cell lysis buffer includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water; wherein the mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is 0.04g:0.03g:0.04g:0.02g:2mL:250mL; the concentration of Tris-HCl is 20mM; the mass volume ratio of the precipitate to the cell lysis buffer is 1g:10mL.

[0122] Example 6

[0123] The embodiment of the present application provides a protein purification partner, including: a universal nuclease and a precipitant, the precipitant is a cationic flocculant, and the cationic flocculant includes polysilicate aluminum iron and polyacrylamide.

[0124] This embodiment also provides a method for using the above protein purification partner, comprising the following steps:

[0125] S1. Obtain Escherichia coli cells;

[0126] S2, adding Escherichia coli cells to the resuspension to obtain a resuspension of cells; the resuspension is a 20 mM Tris-HCl buffer with a pH of 7.5, and the mass volume ratio of the Escherichia coli cells to the resuspension is 1 g:15 mL;

[0127] S3, adjusting the pH of the bacterial resuspension to 8.5, and controlling the temperature to 37°C, then adding universal nuclease for treatment for 1 hour, centrifuging, and collecting the precipitate; wherein, the amount of universal nuclease added is: adding 500U universal nuclease per gram of Escherichia coli;

[0128] S4. Add cell lysis buffer to the precipitate for lysis, and lyse for 1.5 hours. Adjust the pH to 8.5 and control the temperature to 25°C. Then add a polyacrylamide aqueous solution with a mass concentration of 0.1% and a polyaluminum-iron silicate aqueous solution with a mass concentration of 0.1%. For every gram of Escherichia coli cells, add 0.0375 mL of the polyacrylamide aqueous solution and 0.1125 mL of the polyaluminum-iron silicate aqueous solution; the cell lysis buffer comprises Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate and water; wherein the mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl and water is 0.04 g: 0.03 g: 0.04 g: 0.02 g: 2 mL: 250 mL; the concentration of Tris-HCl is 20 mM; the mass volume ratio of the precipitate to the cell lysis buffer is 1 g: 10 mL.

[0129] Comparative Example 5

[0130] This comparative example provides a method for treating Escherichia coli cells, comprising the following steps:

[0131] S1. Obtain Escherichia coli cells;

[0132] S2, adding Escherichia coli cells to the resuspension to obtain a resuspension of cells; the resuspension is a 20 mM Tris-HCl buffer with a pH of 7.5, and the mass volume ratio of the Escherichia coli cells to the resuspension is 1 g:10 mL;

[0133] S3, adjusting the pH of the bacterial resuspension to 8.5, and controlling the temperature to 37°C, then adding universal nuclease for treatment for 1 hour, centrifuging, and collecting the precipitate; wherein, the amount of universal nuclease added is: adding 500U universal nuclease per gram of Escherichia coli;

[0134] S4. Add cell lysis buffer to the precipitate for lysis for 1.5 hours. The cell lysis buffer includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water. The mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is 0.04g:0.03g:0.04g:0.02g:2mL:250mL. The concentration of Tris-HCl is 20mM. The mass volume ratio of precipitate to cell lysis buffer is 1g:10mL.

[0135] Comparative Example 6

[0136] This comparative example provides a method for treating Escherichia coli cells, comprising the following steps:

[0137] S1. Obtain Escherichia coli cells;

[0138] S2, adding Escherichia coli cells to the resuspension to obtain a resuspension of cells; the resuspension is a 20 mM Tris-HCl buffer with a pH of 7.5, and the mass volume ratio of the Escherichia coli cells to the resuspension is 1 g:10 mL;

[0139] S3, adjusting the pH of the bacterial resuspension to 8.5, and controlling the temperature to 37°C, then adding universal nuclease for treatment for 1 hour, centrifuging, and collecting the precipitate; wherein, the amount of universal nuclease added is: adding 50U universal nuclease per gram of Escherichia coli cells;

[0140] S4. Add cell lysis buffer to the precipitate for lysis for 1.5 hours. The cell lysis buffer includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water. The mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is 0.04g:0.03g:0.04g:0.02g:2mL:250mL. The concentration of Tris-HCl is 20mM. The mass volume ratio of precipitate to cell lysis buffer is 1g:5mL.

[0141] Comparative Example 7

[0142] This comparative example provides a method for treating Escherichia coli cells, which is the same as comparative example 6, except that in step S3, 200 U of universal nuclease is added per gram of Escherichia coli cells, and the remaining steps are the same as comparative example 6.

[0143] Comparative Example 8

[0144] This comparative example provides a method for treating Escherichia coli cells, which is the same as comparative example 6, except that in step S3, 500 U of universal nuclease is added per gram of Escherichia coli cells, and the remaining steps are the same as comparative example 6.

[0145] Comparative Example 9

[0146] This comparative example provides a method for treating Escherichia coli cells, which is the same as comparative example 6, except that in step S3, 1000 U of universal nuclease is added per gram of Escherichia coli cells, and the remaining steps are the same as comparative example 6.

[0147] Comparative Example 10

[0148] This comparative example provides a method for treating Escherichia coli cells, which is the same as comparative example 6, except that in step S3, 2000 U of universal nuclease is added per gram of Escherichia coli cells, and the remaining steps are the same as comparative example 6.

[0149] Comparative Example 11

[0150] This comparative example provides a method for treating Escherichia coli cells, which is the same as comparative example 6, except that in step S3, 2500 U of universal nuclease is added per gram of Escherichia coli cells, and the remaining steps are the same as those in comparative example 6.

[0151] According to the methods in Comparative Examples 4 and 6 to 11, the electrophoretic patterns of the supernatant after lysis are as follows: Figure 3 shown.

[0152] Figure 3 In the figure, lane 1: control, corresponding to the corresponding ratio 4, and lanes 2 to 7 correspond to the corresponding ratios 6 to 11, respectively.

[0153] According to the methods in Example 2, Examples 5-6, Comparative Example 5, and Comparative Example 2, the electrophoretic diagram of the supernatant after lysis is as follows: Figure 4 shown.

[0154] Figure 4 In the figure, lane 1: control, corresponding to Example 2, lane 2 corresponds to Example 5, lane 3 corresponds to Example 5, lane 4 corresponds to Example 6, and lane 5 corresponds to Example 2.

[0155] Figures 3-4 In the figure, purification partner A represents the addition of universal nuclease, and purification partner B represents the addition of cationic flocculant.

[0156] from Figures 3-4 It can be seen that with the optimization of the amount of purification partner added, the nucleic acid macromolecules are degraded into small molecules. When the precipitant is used together, the residual nucleic acid amount can be significantly reduced. When 0.05 mL of polyacrylamide aqueous solution and 0.15 mL of polysilicate aluminum ferric aqueous solution are added per gram of Escherichia coli cells in Example 2, and 0.0375 mL of polyacrylamide aqueous solution and 0.1125 mL of polysilicate aluminum ferric aqueous solution are added per gram of Escherichia coli cells in Example 6, there is basically no residual nucleic acid.

[0157] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A protein purification partner, characterized in that include: Universal nuclease and a precipitant, wherein the precipitant is a cationic flocculant.

2. The protein purification partner according to claim 1, characterized in that The cationic flocculant includes at least one of polyacrylamide, polyaluminum ferric silicate, polyaluminum ferric chloride, and polyaluminum phosphate.

3. Use of the protein purification partner as claimed in any one of claims 1 to 2 in the purification of soluble recombinant proteins expressed in bacterial cells.

4. A method for using the protein purification companion according to any one of claims 1 to 2, characterized in that: The following steps are involved: Obtaining bacterial cells; Adding bacterial cells to the resuspension solution to obtain a bacterial resuspension solution; Adding the universal nuclease described in claim 1 to the bacterial resuspension, centrifuging, and collecting the precipitate; Add cell lysis solution to the precipitate for lysis, and then add the precipitant according to any one of claims 1 to 2.

5. The method for using the protein purification companion according to claim 4, characterized in that: The pH of the bacterial resuspension was adjusted to 6.0-11.0, and the temperature was controlled to 0-45°C, and then the universal nuclease was added.

6. The method for using the protein purification companion according to claim 4, characterized in that: Add cell lysis solution to the precipitate for lysis. After lysis is completed, adjust the pH to 6.0-11.0, control the temperature to 2-30° C., and then add the precipitant.

7. The method for using the protein purification companion according to claim 4, characterized in that: Add the universal nuclease described in claim 1 to the bacterial resuspension, treat for 0.5 to 2 hours, centrifuge, and collect the precipitate; wherein the amount of the universal nuclease added is: 20 to 3000 U universal nuclease per gram of bacterial cells; And / or, adding cell lysis solution to the precipitate for lysis, the lysis time is 1 to 12 hours, and then adding the precipitant; wherein the precipitant is added in the form of a precipitant aqueous solution, the mass concentration of the precipitant aqueous solution is 0.1 to 1%, and 0.02 to 0.5 mL of the precipitant aqueous solution is added per gram of bacterial cells.

8. The method for using the protein purification companion according to claim 7, characterized in that: 500 U of universal nuclease was added per gram of bacterial cells, and 0.15-0.2 mL of precipitant aqueous solution was added per gram of bacterial cells.

9. The method for using the protein purification companion according to claim 8, characterized in that: The precipitant aqueous solution comprises a polyacrylamide aqueous solution with a mass concentration of 0.1-1% and a polyaluminum ferric silicate aqueous solution with a mass concentration of 0.1-1%. The volume ratio of the polyacrylamide aqueous solution to the polyaluminum ferric silicate aqueous solution is 1:(2-4).

10. The method for using the protein purification companion according to claim 4, characterized in that: In the step of adding bacterial cells to a resuspension solution, the resuspension solution is a PBS buffer or a Tris-HCl buffer, the pH of the resuspension solution is 7.0-8.0, and the mass volume ratio of the bacterial cells to the resuspension solution is 1 g: (10-20) mL; And / or, the cell lysis solution includes Tris-HCl, EDTA, NaCl, MgCl2, sodium deoxycholate, and water; the mass volume ratio of EDTA, NaCl, MgCl2, sodium deoxycholate, Tris-HCl, and water is (0.04-0.05) g: (0.025-0.035) g: (0.04-0.05) g: (0.02-0.03) g: (1.5-2.0) mL: (200-250) mL; the concentration of Tris-HCl is 18-22 mM; and / or, the mass volume ratio of the precipitate to the cell lysate is (1-2) g:(5-10) mL; And / or, the bacterial cells include Escherichia coli cells or yeast cells.

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