Method for isolating and purifying proteins with at least two histidine tags

Abstract

The present invention relates to a method for isolating and purifying a protein with at least two histidine tags, in particular to a method for isolating and purifying a protein with at least two histidine tags in solution by using cations.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for separating and purifying a protein with at least two histidine tags, in particular to a method for separating and purifying a protein with at least two histidine tags in solution by using cations. BACKGROUND

[0002] Separation and purification of proteins is an important basic operation in the field of biotechnology. There are various protein separation and purification techniques in the prior art, which can be roughly divided into two categories such as chromatography and precipitation.

[0003] Commonly used chromatography methods include immobilized metal ion affinity chromatography (IMAC), immunoaffinity chromatography (IAC), ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), gel filtration chromatography (GFC), etc. Among them, metal ion affinity chromatography (IMAC) is an important protein separation and purification technique in laboratories and industries, which is based on the different binding strengths of metal ions and different proteins at different pH values to achieve protein separation. The use of histidine tags and imidazole makes IMAC mature, and the target protein with high purity (>90%) can be obtained relatively simply and quickly in the laboratory. However, the defects of IMAC are that the stationary phase carrier is expensive, and the transition metal ions used pollute the environment and the target protein. Therefore, IMAC is usually used for protein purification on a laboratory scale or for the purification of high-value-added proteins (such as immunoglobulins).

[0004] A commonly used precipitation method is protein salt precipitation, such as ammonium sulfate precipitation. Salt precipitation is a method of precipitating proteins by adding a large amount of neutral salt (such as ammonium sulfate, the concentration of ammonium sulfate in the salt precipitation system is as high as about 2 mol / L or higher) to the protein solution. The characteristics of this method are low cost, better stability of the precipitated protein, and better storage and transportation, but the purity of the obtained protein is not ideal, and a large amount of salt is mixed in the precipitate, which may affect subsequent use. The wastewater containing high-concentration ammonia nitrogen also has adverse effects on the environment.

[0005] So far, ammonium sulfate precipitation and IMAC are still the two main protein purification methods, but each has its own shortcomings. Researchers in the field have been trying to find a more ideal purification method that combines the advantages of both. SUMMARY

[0006] The present inventors have found, through extensive research, that a protein with at least two histidine tags (also referred to herein as a target protein) can be selectively precipitated when contacted with a very low concentration of cations in an aqueous solution, and the precipitate mainly contains the target protein, while most of the impurities remain in the supernatant.

[0007] In addition, the inventors of the present application have surprisingly found that the target protein selectively precipitated can be re-dissolved in an aqueous solution when contacted with a specific concentration of cations, while the precipitated impurities are relatively less dissolved.

[0008] Meanwhile, the inventors of the present application have surprisingly found that when a mixture containing the target protein and the impurities is contacted with a specific concentration of cations in an aqueous solution, the impurities can be selectively precipitated relative to the target protein.

[0009] That is, by selecting appropriate cations and their concentrations, the target protein can be selectively precipitated and selectively dissolved, thereby achieving separation and purification of the target protein.

[0010] The method of the present application has the advantages of not requiring expensive carriers and complex operating conditions, using very low concentrations of cations, being environmentally friendly, being simple to operate, having very low costs, and having very good protein purification results (comparable or better than IMAC, but at much lower costs than IMAC). BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1a The solubility of red fluorescent protein with and without a histidine tag in a NaCl system at different NaCl concentrations is shown. + The solubility of red fluorescent protein with and without a histidine tag in a Na2SO4 system at different Na2SO4 concentrations is shown.

[0012] Figure 1b The solubility of red fluorescent protein with and without a histidine tag in a NaCl system at different NaCl concentrations is shown. + The solubility of red fluorescent protein with and without a histidine tag in a Na2SO4 system at different Na2SO4 concentrations is shown.

[0013] Figure 1c The solubility of red fluorescent protein with and without a histidine tag in a NaCl system at different NaCl concentrations is shown. + The solubility of red fluorescent protein with and without a histidine tag in a Na2SO4 system at different Na2SO4 concentrations is shown.

[0014] Figure 1d The solubility of red fluorescent protein with and without a histidine tag in a NaCl system at different NaCl concentrations is shown. + The solubility of red fluorescent protein with and without a histidine tag in a Na2SO4 system at different Na2SO4 concentrations is shown.

[0015] Figure 1e The solubility of red fluorescent protein with and without a histidine tag in a NaCl system at different NaCl concentrations is shown. 4+ The solubility of red fluorescent protein with and without a histidine tag in a Na2SO4 system at different Na2SO4 concentrations is shown.

[0016] Figure 1f The solubility of red fluorescent protein with and without a histidine tag in a NaCl system at different NaCl concentrations is shown. 4+ The solubility of red fluorescent protein with and without a histidine tag in a Na2SO4 system at different Na2SO4 concentrations is shown.

[0017] Figure 1g Solubility of red fluorescent protein with and without histidine tag in different lysine concentrations in the system is shown.

[0018] Figure 1h Solubility of red fluorescent protein with and without histidine tag in different imidazole concentrations in the system is shown.

[0019] Figure 1i Solubility of red fluorescent protein with and without histidine tag in different guanidine hydrochloride concentrations in the system is shown.

[0020] Figure 1j Solubility of red fluorescent protein with and without histidine tag in different urea concentrations in the system is shown.

[0021] Figure 2a Solubility of red fluorescent protein with and without histidine tag in different Mg 2+ concentrations in MgCl2 system is shown.

[0022] Figure 2b Solubility of red fluorescent protein with and without histidine tag in different Mg 2+ concentrations in MgSO4 system is shown.

[0023] Figure 2c Solubility of red fluorescent protein with and without histidine tag in different Ca 2+ concentrations in the system is shown.

[0024] Figure 2d Solubility of red fluorescent protein with and without histidine tag in different Mn 2+ concentrations in the system is shown.

[0025] Figure 2e Solubility of red fluorescent protein with and without histidine tag in different Ni 2+ concentrations in the system is shown.

[0026] Figure 2f Solubility of red fluorescent protein with and without histidine tag in different Zn 2+ concentrations in the system is shown.

[0027] Figure 2g Solubility of red fluorescent protein with and without histidine tag in different Cu 2+ concentrations in the system is shown.

[0028] Figure 3a Photograph of the precipitation results of red fluorescent protein with histidine tag treated with different divalent cations at 2mM.

[0029] Figure 3b Protein electrophoresis results of the precipitate and supernatant samples of red fluorescent protein with histidine tag treated with different divalent cations at 2mM.

[0030] Figure 4 Protein electrophoresis results of the precipitate and supernatant samples of red fluorescent protein with histidine tag treated with different concentrations of Mg 2+ Results of the resuspension of the precipitate containing red fluorescent protein with histidine tag.

[0031] Figure 5 Protein electrophoresis results of the precipitate and supernatant samples of L-threonine aldolase with histidine tag treated with different concentrations of magnesium ions.

[0032] Figure 6 Protein electrophoresis results of the precipitate and supernatant samples of L-threonine aldolase with histidine tag treated with Mg 2+ Results of the precipitation, resuspension and re-precipitation of L-threonine aldolase with histidine tag.

[0033] Figure 7 Protein electrophoresis results of the precipitate and supernatant samples of L-threonine aldolase with histidine tag treated with Mg 2+ Results of the precipitation, resuspension and re-precipitation of formate dehydrogenase with histidine tag.

[0034] Figure 8 Protein electrophoresis results of the precipitate and supernatant samples of L-threonine aldolase with histidine tag treated with Mg 2+ Results of the precipitation, resuspension and re-precipitation of L-leucine dehydrogenase with histidine tag.

[0035] Figure 9 Results of the precipitation, resuspension and re-precipitation of amino acid ester acyltransferase with histidine tag.

[0036] Figure 10 Protein electrophoresis results of the supernatant and precipitate samples of ND treated with different pH of sodium phosphate buffer.

[0037] Figure 11 Supernatant fluorescence results of ND treated with Mg 2+ at different total protein concentrations.

[0038] Figure 12 Supernatant and precipitate results of green fluorescent protein with different histidine tags treated with magnesium ions at 10mM concentration.

[0039] Figure 13 Supernatant and precipitate results of pyrophosphothiamine kinase with 2 histidine tags treated with potassium, sodium, ammonium ions at 100mM concentration.

[0040] Figure 14 Supernatant and pellet results of polyphosphate kinase with 2 histidine tags after treatment with potassium, sodium, ammonium ions at 100 mM concentration.

[0041] Figure 15 Protein electrophoresis results of L-leucine dehydrogenase with histidine tag after purification by magnesium, sodium, ammonium ion precipitation and IMAC.

[0042] Figure 16 Protein electrophoresis results of red fluorescent protein with histidine tag after purification by magnesium, sodium, ammonium ion precipitation and IMAC.

[0043] Figure 17 Protein electrophoresis results of L-threonine aldolase with histidine tag after purification by magnesium, sodium, ammonium ion precipitation and IMAC.

[0044] Figure 18 Electrophoresis results of ND precipitated and resuspended with different ions. DETAILED DESCRIPTION

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In case of conflict, the present specification, including definitions, will control.

[0046] When expressing a quantity, concentration or other value or parameter either as a range, a preferred range, or a range preferred or exemplary, it is intended to include every individual value, sub-range, and whole and part- fraction within the range. Unless otherwise stated, the numerical values listed in the specification are intended to be approximations.

[0047] The materials, methods, and examples of the application are illustrative only and not intended to be limiting.

[0048] When the term "about" is used in connection with a value or range of values, it is intended to encompass the ±5%, preferably ±3%, more preferably ±1% range of the referenced value or endpoint. In the present application, unless otherwise indicated, all numbers expressing quantities, concentrations, and other values are to be understood as modified in all instances by the term "about".

[0049] Definitions

[0050] In the present text, the term "histidine tag" refers to a short peptide consisting of a string of consecutive histidine residues, for example a short peptide consisting of 3 to 15 consecutive histidine residues, in particular a short peptide consisting of 3, 4, 5, 6, 8, 9, 10 or 12 consecutive histidine residues.

[0051] The expression "protein with at least two histidine tags" refers to a protein with at least two of the above "histidine tags" not directly linked to each other, that is, with two short peptides consisting of a string of consecutive histidine residues and the two short peptides are not directly linked to each other.

[0052] In case the protein consists of a single protein subunit (also referred to herein as monomeric protein or monomeric protein), one "histidine tag" can be present at each end of the protein subunit. In case the protein consists of more than two identical protein subunits (also referred to herein as multimeric protein or multimeric protein), one "histidine tag" can be present at one or both ends of each protein subunit.

[0053] In the present text, the expression "protein with at least two histidine tags" has the same meaning as the term "protein of interest" and can be used interchangeably.

[0054] The "protein with at least two histidine tags" in the present text can be obtained by methods conventional in the art, for example by recombinant gene expression.

[0055] In the present text, the term "aqueous solution" refers to a solution system in which the solvent is predominantly water, and in which solution system other solvents which are unavoidable in the raw materials can be contained. By "predominantly water" is meant that 95% by weight of the total amount of solvent is water, preferably 97% by weight of the solvent is water, more preferably 99% by weight of the solvent is water. Preferably, the "aqueous solution" refers to a solution system in which the solvent is water.

[0056] In the present text, the term "non-H + " refers to monovalent cations other than H + , preferably selected from one or more of NH4 + , Na + , K + and organic nitrogen-containing cations.

[0057] In the present context, the term "organic nitrogen-containing cation" is those cations in which the nitrogen is in the tetravalent state and at least one group attached to N is an organic group. Preferably, the "organic nitrogen-containing cation" is derived from an organic molecule, preferably an organic small molecule, containing -NH2, -NH- and / or =N- groups. The "organic small molecule" refers to an organic molecule in which there are no repeating polymeric units present in the molecule. More preferably, the organic nitrogen-containing cation is derived from imidazole, glucosamine, guanidine hydrochloride, lysine, arginine and histidine.

[0058] In the present context, the term "heteroprotein" refers to a protein different from the protein of interest.

[0059] The following describes a specific embodiment of the method of the present application.

[0060] In a first aspect of the present application, there is provided a method for isolating and purifying a protein carrying at least two histidine tags, said method comprising: contacting said protein in a solubilized state with a cation in an aqueous solution, to obtain a precipitate comprising said protein;

[0061] wherein the pH of the aqueous solution is in the range of 7.2 or less, preferably in the range of 5.0-7.0, more preferably in the range of 6.0-7.0;

[0062] said cation is selected from one or more of the following: non-H + monovalent cations, Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ ;

[0063] when said cation is selected from non-H + monovalent cations, the concentration of said cation in the aqueous solution is in the range of 10-400 mmol / L, preferably in the range of 15-300 mmol / L, more preferably in the range of 30-200 mmol / L;

[0064] when said cation is selected from Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ , the concentration of said cation in the aqueous solution is in the range of 1-80 mmol / L, preferably in the range of 1.5-40 mmol / L, more preferably in the range of 3-30 mmol / L;

[0065] In the method of the first aspect, the expression "contacting said protein in a solubilized state with a cation in an aqueous solution" can be carried out, for example, by the following method:

[0066] - dissolving the mixture containing the target protein in water to form a crude protein solution, and then adding the water-soluble salt containing the cation (in solid or aqueous solution form) to the crude protein solution;

[0067] - dissolving the water-soluble salt containing the cation in water to form a salt solution, and then adding the mixture containing the target protein (in solid or aqueous solution form) to the salt solution; and / or

[0068] - dissolving the water-soluble salt containing the cation in water to form a salt solution, suspending the bacterial cells containing the target protein in the salt solution, and then disrupting the bacterial cells in the salt solution and releasing the target protein into the salt solution.

[0069] The above-mentioned methods are merely exemplary, and one skilled in the art can use other methods to contact the target protein in a dissolved state with the cation in an aqueous solution as needed for experiments.

[0070] In the method of the first aspect, the pH of the aqueous solution should be maintained in the range of 7.2 or less. When the pH of the system (which can refer to the aqueous solution) is greater than 7.2, the target protein cannot be effectively precipitated. From the perspective of better selectively precipitating the target protein, it is preferable to set the pH of the system to be not less than 5.0. For example, the pH can be set to 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1.

[0071] In the method of the first aspect, the cation is selected from one or more of the following: monovalent cations other than H + +, Mg 2 + +, Ca 2+ +, Mn 2+ +, Fe 2+ +, and Co 2+ +. The cation can be used in only one kind, or two or more kinds in combination. From the perspective of industrial usability, cost reduction, or pollution reduction, it can be preferable to use a cation selected from NH4 + +, Na + +, K + +, Mg 2+ +, and Ca 2+ +.

[0072] In the prior art of IMAC, the purification of a protein is achieved by binding the protein to be separated to metal ions immobilized on a carrier, and then eluting. The principle of purification of IMAC teaches the binding of the protein to be separated to metal ions on a carrier, but the binding on the carrier does not necessarily mean that the protein to be separated can be precipitated from the solution using metal ions. That is, the binding on the carrier and the direct precipitation separation from the solution are two different concepts.

[0073] In addition, nickel ions, copper ions and zinc ions commonly used in IMAC can precipitate the target protein in an aqueous solution system, but at the same time, almost all the impurity proteins are precipitated without distinction. Therefore, cations such as nickel ions, copper ions and zinc ions cannot achieve selective precipitation of the target protein. In addition, IMAC does not use monovalent cations for separation.

[0074] In the method of the first aspect, when the cation is selected from non-H + monovalent cations, the concentration of the cation in the aqueous solution is 10-400 mmol / L, preferably 15-300 mmol / L, more preferably 30-200 mmol / L, for example 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 55, 60, 70, 85, 90, 100, 110, 125, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 255, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 mmol / L; when the cation is selected from Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ and Co 2+ , the concentration of the cation in the aqueous solution is 1-80 mmol / L, preferably 1.5-40 mmol / L, more preferably 3-30 mmol / L, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 45, 46, 47, 48, 49, 50, 52, 55, 57, 58, 60, 63, 65, 67, 70, 72, 75, 78 mmol / L;

[0075] The ammonium sulfate precipitation method, for example, generally uses a concentration of about 2 mol / L or more of ammonium sulfate in a solution, and thus, in comparison with the ammonium sulfate precipitation method, the method of the present application can achieve the precipitation of the target protein using a much lower concentration of salt. Moreover, the above-mentioned concentration of the method of the present application can achieve the selective precipitation of the target protein.

[0076] In addition, the total protein concentration has substantially no effect on the method of the present application.

[0077] In the method of the first application, the selective precipitation of the target protein is achieved mainly by selecting the kind and concentration of the cation in combination with a specific pH.

[0078] As described above, the inventors of the present application have also surprisingly found that the target protein that is selectively precipitated can be re-dissolved when contacted with a specific concentration of a cation in an aqueous solution, while the precipitated impurities are relatively less dissolved. That is, the precipitated target protein can be selectively dissolved.

[0079] Thus, in a second aspect of the present application, there is provided a method for isolating and purifying a protein having at least two histidine tags, the method comprising:

[0080] a) contacting the protein in a dissolved state with a cation at a first concentration in an aqueous solution to obtain a precipitate comprising the protein;

[0081] b) separating the precipitate from the supernatant;

[0082] c) contacting the separated precipitate with a cation at a second concentration in an aqueous solution, so that at least a portion of the protein contained in the precipitate is re-dissolved;

[0083] wherein the pH of the aqueous solution in step a) and step c) is each independently in the range of 7.2 or less, preferably 5.0-7.0, more preferably in the range of 6.0-7.0;

[0084] the cation in step a) and step c) is each independently selected from one or more of the following: a monovalent cation other than H + , Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ ;

[0085] In step a), when the cation is selected from a monovalent cation other than H +1.5-40 mmol / L, more preferably 3-30 mmol / L, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 mmol / L, when the cation is selected from the group consisting of Mg 2+ ,Ca 2+ ,Mn 2+ ,Fe 2+ and Co 2+ ;

[0086] In step c), when the cation is selected from monovalent cations other than H + +, the second concentration is equal to or greater than 250 mmol / L, preferably equal to or greater than 400 mmol / L, more preferably equal to or greater than 600 mmol / L, but not more than 1000 mmol / L, more preferably not more than 800 mmol / L, for example 251, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 820, 940, 960 mmol / L, or the second concentration is equal to or less than 30 mmol / L, preferably equal to or less than 20 mmol / L, more preferably equal to or less than 10 mmol / L, for example 29, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 0 mmol / L;

[0087] In step c), when the cation is selected from Mg 2+ ,Ca 2+ ,Mn 2+ ,Fe 2+ and Co2+ In some embodiments, the second concentration is greater than or equal to 35 mmol / L, preferably greater than or equal to 50 mmol / L, more preferably greater than or equal to 100 mmol / L, but not more than 250 mmol / L, more preferably not more than 200 mmol / L, for example 36, 38, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240 mmol / L; or, the second concentration is less than or equal to 2 mmol / L, preferably less than or equal to 1 mmol / L, for example 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0 mmol / L.

[0088] In the method of the second aspect, examples of implementation of the expression "contacting the protein in a dissolved state with a cation at a first concentration in an aqueous solution" in step a) can refer to the applicable examples of implementation of the expression "contacting the protein in a dissolved state with a cation in an aqueous solution" in the method of the first aspect. Similarly, these implementation methods are only exemplary, and one skilled in the art can use other ways to contact the target protein in a dissolved state with a cation at a first concentration in an aqueous solution according to experimental needs.

[0089] For the definition of pH in steps a) and c) in the method of the second aspect, the expression of pH in the method of the first aspect can be applied.

[0090] For the selection of cation species in steps a) and c) in the method of the second aspect, the expression of the selection of cation species in the method of the first aspect can be similarly applied.

[0091] In fact, the relationship between the concentration of the target protein in the aqueous solution and the concentration of the cation in the aqueous solution is a U-shaped curve in the X_Y coordinate system with the concentration of the target protein as the vertical coordinate Y and the concentration of the cation as the horizontal coordinate X. That is, the concentration of the target protein in the aqueous solution continuously decreases with the increase of the concentration of the cation in the first stage (i.e. the left side of the lowest point of the U-shaped curve), the target protein continuously precipitates out, and at a certain concentration of the cation, the lowest point of the U-shaped curve is reached (i.e. the maximum amount of target protein is precipitated out), then with the increase of the concentration of the cation, the target protein begins to dissolve, and after a certain concentration of the cation, the target protein is completely dissolved.

[0092] In step a) where the target protein is precipitated, the cation concentration that can cause the target protein to precipitate includes most of the cation concentrations on the horizontal axis of the U-shaped curve, the only difference being the amount of target protein precipitated. However, from the viewpoint of the economy and practicality of industrial operation, it is preferred that as much target protein as possible be precipitated in step a), and therefore, in step a), when the cation is selected from monovalent cations other than H + , the first concentration is 15 to 300 mmol / L, preferably 30 to 200 mmol / L, more preferably 80 to 120 mmol / L; when the cation is selected from Mg 2+ , Ca 2+ , Fe 2+ , Mn 2+ and Co 2+ , the first concentration is 1.5 to 40 mmol / L, more preferably 3 to 30 mmol / L.

[0093] For step b) of the method of the second aspect, the precipitate is separated from the supernatant by a conventional solid-liquid separation method, and there is no particular limitation, for example, centrifugal separation.

[0094] For step c) of the method of the second aspect, for "contacting the separated precipitate with a second concentration of cations in an aqueous solution", it can be performed by any suitable means, for example, by adding the precipitate to an aqueous solution containing cations or by adding an aqueous solution of cations to the precipitate so that the final concentration of cations in the aqueous solution falls within the range of the second concentration.

[0095] In step c) where the target protein in the precipitate is dissolved, according to the solubility U-shaped curve, the purpose of redissolving the precipitated target protein can be achieved as long as it is on the left or right side of the lowest point of the U-shaped curve. However, from the viewpoint of the economy of process operation, it is preferred that in step c), when the cation is selected from monovalent cations other than H + , the second concentration is equal to or greater than 250 mmol / L, preferably equal to or greater than 400 mmol / L, more preferably equal to or greater than 600 mmol / L, but not more than 1000 mmol / L, more preferably not more than 800 mmol / L, or the second concentration is equal to or less than 30 mmol / L, preferably equal to or less than 20 mmol / L, more preferably equal to or less than 10 mmol / L; in step c), when the cation is selected from Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ and Co 2+When the second concentration is greater than or equal to 35 mmol / L, preferably greater than or equal to 50 mmol / L, more preferably greater than or equal to 100 mmol / L, but not exceeding 250 mmol / L, more preferably not exceeding 200 mmol / L, or the second concentration is less than or equal to 2 mmol / L, preferably less than or equal to 1 mmol / L. The second concentration can be 0 mmol / L.

[0096] In step c), the concentration of cations used for redissolution should not be too high, as this can cause protein salting-out precipitation. As mentioned earlier, after the precipitate comes into contact with a second concentration of cations, the target protein can be redissolved, while less of the other proteins dissolve. Therefore, the target protein can undergo a precipitation-resolution-reprecipitation process for continuous purification. The precipitation-resolution step can be repeated multiple times as needed until the desired target protein purity is achieved.

[0097] Therefore, the method of the second aspect of this application may further include step d): separating the supernatant obtained in step c) and adjusting the cation concentration therein to the first concentration range, so that at least a portion of the protein in the supernatant is reprecipitated.

[0098] As mentioned earlier, when a mixture containing the target protein and other proteins is contacted with a certain concentration of cations in an aqueous solution, the other proteins can be selectively precipitated relative to the target protein.

[0099] Therefore, in a third aspect of the invention, a method for isolating and purifying proteins with at least two histidine tags is provided, the method comprising:

[0100] a) Contact the mixture of the protein and other proteins in a dissolved state with a third concentration of cations in an aqueous solution to obtain a precipitate containing the other proteins and a supernatant containing the proteins.

[0101] b) Separate the precipitate from the supernatant; and

[0102] c) Dilute the separated supernatant to a fourth concentration of cations, such that at least a portion of the protein contained in the supernatant precipitates;

[0103] Wherein, the pH value of the aqueous solution in steps a) and c) is independently within the range of less than or equal to 7.2, preferably 5.0-7.0, and more preferably within the range of 6.0-7.0;

[0104] The cations in steps a) and c) are each independently selected from one or more of the following: non-H + Monovalent cation, Mg 2+ Ca2+ Mn 2+ Fe 2+ and Co 2+ ;

[0105] In step a), when the cation is selected from monovalent cations other than H + +, the third concentration is greater than or equal to 250 mmol / L, preferably greater than or equal to 400 mmol / L, more preferably greater than or equal to 600 mmol / L, but not more than 1000 mmol / L, more preferably not more than 800 mmol / L, for example 251, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 820, 940, 960 mmol / L; when the cation is selected from Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ and Co 2+ , the third concentration is greater than or equal to 35 mmol / L, preferably greater than or equal to 50 mmol / L, more preferably greater than or equal to 100 mmol / L, but not more than 250 mmol / L, more preferably not more than 200 mmol / L, for example 36, 38, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240 mmol / L.

[0106] In step c), when the cation is selected from monovalent cations other than H + + the third concentration is greater than or equal to 250 mmol / L, preferably greater than or equal to 400 mmol / L, more preferably greater than or equal to 600 mmol / L, but not more than 1000 mmol / L, more preferably not more than 800 mmol / L, for example 251, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 820, 940, 960 mmol / L; when the cation is selected from Mgwhen the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ and Co 2+ , the fourth concentration is 1.5-40 mmol / L, more preferably 3-30 mmol / L, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 mmol / L.

[0107] In the method of the third aspect, the manner of "contacting" in step a) is with reference to the manner of "contacting" in the method of the first aspect.

[0108] In the method of the third aspect, the pH in steps a) and c) can be with reference to the expression of pH in the method of the first aspect.

[0109] In the method of the third aspect, the selection of the kind of cation in steps a) and c) can be with reference to the expression of the selection of the kind of cation in the method of the first aspect.

[0110] In the method of the third aspect, the meaning of step a) is to use a cation concentration higher than the optimal precipitation point, so that the impurity proteins can be selectively precipitated out, thereby at least partially purifying the target proteins in the supernatant.

[0111] For step b) in the method of the third aspect, the precipitate is separated from the supernatant, which can be performed by using conventional solid-liquid separation methods without particular limitations, such as centrifugal separation.

[0112] In the method of the third aspect, step c) is to precipitate the target protein by reducing the concentration of cations for the target protein which has been at least partially purified in the supernatant. As long as the fourth concentration in step c) is lower than the third concentration, at least a part of the target protein can be precipitated, and the most target protein can be precipitated when the concentration of cations is reduced to the concentration at the lowest end of the U-shaped curve. However, from the viewpoint of the economy and practicability of industrial operation, it is preferred that, when the cations are selected from monovalent cations other than H + , the fourth concentration is 15-300 mmol / L, preferably 30-200 mmol / L, and more preferably 80-120 mmol / L; and when the cations are selected from Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ and Co 2+ , the fourth concentration is 1.5-40 mmol / L, and more preferably 3-30 mmol / L.

[0113] In the method of the third aspect, after step c), a further step d) can be included, i.e. separating the precipitate obtained in step c) and contacting the precipitate with cations at a second concentration in an aqueous solution, so that at least a part of the protein contained in the precipitate is redissolved; here the second concentration refers to the second concentration described in the method of the second aspect.

[0114] For the specific mode of step d) in the method of the third aspect, refer to the description of step c) in the method of the second aspect.

[0115] For the method of the third aspect, after the target protein is precipitated, according to the actual needs, the redissolution-precipitation can be repeated for multiple times until the desired purity of the target protein is obtained.

[0116] Examples The technical solutions of the present application are further described below in combination with specific examples, but the present application is not limited to the following examples.

[0117] Materials used

[0118] Red fluorescent protein with histidine tag (4 subunits, 4 tags) Red fluorescent protein with histidine tag (6 histidine residues) at the N-terminal of the subunit of the expressed protein (N-his-DsRed, referred to as ND) is cloned and expressed using E. coli pET28a plasmid as the vector. The red fluorescent protein has a total of 4 subunits, and therefore a total of 4 histidine tags.

[0119] - Red fluorescent protein without tag (4 subunits, 0 tags) was expressed with no histidine tag using E. coli pET28a plasmid as the vector (Wild-type DsRed, abbreviated as WD).

[0120] - L-leucine dehydrogenase with histidine tag (8 subunits, 8 tags) was expressed with a histidine tag at the N-terminus of each subunit (6 histidine residues) using E. coli pET28a plasmid as the vector. There are 8 subunits of L-leucine dehydrogenase, so there are 8 histidine tags in total.

[0121] - Formate dehydrogenase with histidine tag (2 subunits, 2 tags) was expressed with a histidine tag at the N-terminus of each subunit (6 histidine residues) using E. coli pET28a plasmid as the vector. There are 2 subunits of formate dehydrogenase, so there are 2 histidine tags in total.

[0122] - Amino acid ester acyltransferase with histidine tag (2 subunits, 2 tags) was expressed with a histidine tag at the N-terminus of each subunit (6 histidine residues) using E. coli pET28a plasmid as the vector. There are 2 subunits of amino acid ester acyltransferase, so there are 2 histidine tags in total.

[0123] - L-threonine aldolase with histidine tag (4 subunits, 4 tags) was expressed with a histidine tag at the N-terminus of each subunit (6 histidine residues) using E. coli pET28a plasmid as the vector. There are 4 subunits of L-threonine aldolase, so there are 4 histidine tags in total.

[0124] - Green fluorescent protein with two histidine tags (single subunit, 2 tags) was expressed with a histidine tag at the N-terminus and C-terminus of the subunit (6 histidine residues) using E. coli pET28a plasmid as the vector (Double-his-GFP, abbreviated as DG). There is only 1 subunit of green fluorescent protein, but it has 2 histidine tags.

[0125] - Green fluorescent protein with a single histidine tag (single subunit, 1 tag) was expressed with a histidine tag at the N-terminus or C-terminus of the subunit (6 histidine residues) using E. coli pET28a plasmid as the vector (N-his-GFP or C-his-GFP, abbreviated as NG or CG). There is only 1 subunit of green fluorescent protein, and it has 1 histidine tag.

[0126] - Unlabeled green fluorescent protein (single subunit, 0 tags) was expressed using E. coli pET28a plasmid as a vector to clone the green fluorescent protein without a fused histidine tag (Wildtype GFP, abbreviated as WG).

[0127] - Pyruvate phosphotransferase kinase with two histidine tags (single subunit, 2 tags) was expressed using E. coli pET28a plasmid as a vector to clone the protein subunit with a histidine tag (6 histidine residues) at the N- and C-termini. Pyruvate phosphotransferase kinase has only one subunit, but has two histidine tags.

[0128] - Pyruvate phosphotransferase kinase with two histidine tags (single subunit, 2 tags) was expressed using E. coli pET28a plasmid as a vector to clone the protein subunit with a histidine tag (6 histidine residues) at the N- and C-termini. Pyruvate phosphotransferase kinase has only one subunit, but has two histidine tags.

[0129] Methods involved

[0130] 1. Bacterial culture

[0131] Inoculate 1‰ volume of kanamycin and 1% volume of glycerol bacteria into LB liquid medium (10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl), incubate at 37°C, 200 rpm for 14 h, inoculate the bacterial liquid into liquid medium containing 1‰ kanamycin (10 g / L of proteose peptone, 5 g / L of yeast powder, 8.95 g / L of Na2HPO4, 3.4 g / L of KH2PO4, 2.67 g / L of NH4Cl, 0.7 g / L of Na2SO4, 0.24 g / L of MgSO4, 5 g / L of glycerol, 0.5 g / L of glucose, 2 g / L of lactose) at a medium volume of 2.5%, incubate at 28°C, 200 rpm for 24 h, and centrifuge to harvest the bacterial cells.

[0132] 2. Total protein concentration determination

[0133] The total protein concentration determination was performed using BCA colorimetric method according to the instructions of the BCA protein concentration determination kit of Beijing Solabio Science and Technology Co., Ltd.

[0134] 3. Red fluorescent protein fluorescence determination

[0135] (1) Dilute the sample to an appropriate concentration,

[0136] (2) Take 100 μL of enzyme solution in a 96-well plate, and then use an enzyme marker to detect the fluorescence absorption at 591 nm under an excitation wavelength of 554 nm.

[0137] 4. Green fluorescent protein fluorescence determination

[0138] (1) Dilute the sample to a suitable concentration,

[0139] (2) Take 100 μL enzyme solution in a 96-well plate, then detect the fluorescence absorption at 520 nm under 488 nm excitation wavelength with a microplate reader.

[0140] 5. L-leucine dehydrogenase enzyme activity assay

[0141] (1) Prepare the substrate solution: prepare a substrate solution containing 4.5 mM 2-oxobutyrate and 0.204 mM NADH with 900 mM NH3-NH4Cl buffer at pH = 9.5.

[0142] (2) Take 20 μL enzyme solution in a 96-well plate, quickly add 180 μL substrate and mix, then immediately detect the absorbance change at 340 nm over time.

[0143] (3) Calculate the NADH concentration change value from the absorbance, and calculate the enzyme activity from the NADH concentration change value and time. The enzyme activity is defined as 1 U, which is the amount of enzyme required to catalyze the consumption of 1 μmol NADH per minute.

[0144] 6. L-threonine aldolase enzyme activity assay

[0145] (1) Prepare the substrate solution: prepare a solution containing 40 mM DL-phenylserine, 40 μM pyridoxal phosphate (PLP) with deionized water, and finally adjust the pH to 8.5.

[0146] (2) Take 10 μL enzyme solution in a centrifuge tube, add 190 μL deionized water and 200 μL substrate and mix, react at 30°C for 10 min, and then stop with 400 μL 1.7% phosphoric acid stop solution. Then detect the absorbance value at 290 nm.

[0147] (3) Calculate the product benzaldehyde concentration change value from the absorbance and standard curve, and calculate the enzyme activity from the benzaldehyde concentration change value and time. The enzyme activity is defined as 1 U, which is the amount of enzyme required to catalyze the production of 1 μmol benzaldehyde per minute.

[0148] 7. Formate dehydrogenase enzyme activity assay

[0149] (1) Prepare the substrate solution: prepare a solution containing 167 mM sodium formate, 100 mM β-mercaptoethanol, 1.67 mM NAD+ with 0.1 M phosphate buffer at pH = 7.5.

[0150] (2) Take 20 μL enzyme solution in a 96-well plate, quickly add 180 μL substrate and mix, then immediately detect the absorbance change at 340 nm over time.

[0151] (3) Calculate the NADH concentration change value based on absorbance and standard curve, and calculate enzyme activity based on the NADH concentration change value and time. Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol NADH per minute, defined as 1 U.

[0152] Example 1: Selective precipitation of red fluorescent protein with histidine tag using monovalent cations

[0153] Using the E. coli pET28a plasmid as a vector, the four-subunit red fluorescent protein (ND) with a histidine tag (6 histidine residues) at the N-terminus of the protein subunit was cloned and expressed (with a total of 4 histidine tags). The obtained bacterial cells were resuspended in deionized water, and crude protein solution 1 was obtained after the bacterial cells were broken.

[0154] The tetrasubunit red fluorescent protein (WD) without histidine tag was cloned and expressed using the E. coli pET28a plasmid as a vector. The obtained bacterial cells were resuspended in deionized water and the crude protein solution 2 was obtained after the bacterial cells were broken.

[0155] Take 500 μl of crude protein solution 1, and add different volumes of 2 mol / L NaCl aqueous solution and deionized water to it respectively, so that the total volume of the system is 1 mL, and the final Na content in the system is... + Concentrations of 0, 0.1, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 400, 600, 800, and 1000 mmol / L were used, with a pH of 6.2. The mixture was incubated at 4°C for 1 hour, followed by centrifugation at 12000 rpm for 2 minutes. The fluorescence absorption of the supernatant at 591 nm under a 554 nm excitation wavelength was measured using a microplate reader to determine the proportion of residual fluorescence in the solution. By comparing the measured proportion of residual fluorescence in the supernatant, the proportion of red fluorescent protein remaining in the supernatant can be determined.

[0156] Parallel to the operation of crude protein solution 1 described above, similar operations were performed using crude protein solution 2 (WD, control).

[0157] The results show that the ratio of residual fluorescence in the supernatant to the fluorescence of the original sample is used as the ordinate, and the Na+ content in the system is used as the plotting factor. + Plotting the concentration (mM) on a logarithmic scale with the x-axis as the horizontal axis, as shown below. Figure 1a As shown. By Figure 1a As can be seen, when Na + At concentrations ranging from 10 to 400 mmol / L (mM), histidine-tagged ND achieved selective precipitation compared to untagged WD.

[0158] Similar to the operation with NaCl, the NaCl is replaced with Na2SO4 (Figure 1b ), LiCl ( Figure 1c ), KCl ( Figure 1d ), NH4Cl ( Figure 1e ), (NH4)2SO4 ( Figure 1f ), lysine ( Figure 1g ), imidazole ( Figure 1h ), and guanidine hydrochloride ( Figure 1i ). It can be seen that when the concentration of these monovalent cations is in the range of about 10 to about 400 mM, especially in the range of 15-300 mM, the ND with histidine tag is selectively precipitated compared with the WD without tag. Figures 1b to 1i

[0159] In addition, similar to the experiment with NaCl, using a system with a concentration of 100 mM of histidine cation or glucosamine cation or arginine, it was found that 95% or 97% or 97.5% of the ND with histidine tag was precipitated, while the WD without histidine tag hardly precipitated.

[0160] As a comparative example, similar to the operation of NaCl, NaCl was changed to urea (NH2-C(=O)-NH2) respectively, and the results are shown in Figure 1j Since urea does not dissociate to produce cations in aqueous solution, it cannot cause selective precipitation of the target protein.

[0161] In addition, taking NH4Cl as an example, the yield and purity of the one-step precipitation of ND obtained at different concentrations were investigated. 500 μL of ND was taken, 500 μL of NH4Cl solution was added, so that the corresponding final concentration of NH4 + was 50, 100, 200 mM respectively, the pH value of the mixed sample was 6.2, and the precipitation was carried out at 4°C for 1 h. Centrifugation at 12000 rpm for 2 min, detection of fluorescence and protein concentration in supernatant, calculation of one-step precipitation yield and purity of ND. The purity was calculated in the form of specific fluorescence.

[0162] Precipitation yield = (original fluorescence - supernatant residual fluorescence) / original fluorescence

[0163] Specific fluorescence = fluorescence of sample / protein concentration of sample

[0164] Purification fold = specific fluorescence of sample after purification / specific fluorescence of crude protein solution

[0165] The results are shown in Table 1 below.

[0166] Table 1: NH4Cl one-step precipitation yield and purity of ND

[0167]

[0168] As Figure 1d ​and 1f As shown in Table 1, the WD did not produce a precipitate when the above concentrations of NH4Cl were added, while the target protein ND, as shown in Table 1, did produce a precipitate when the above concentrations of NH4Cl were added. + The target protein can be selectively precipitated, and the yield is high, and the purity is greatly improved.

[0169] As can be seen from the results of Example 1, the non-H + The monovalent cations can selectively precipitate the red fluorescent protein with a histidine tag in a certain concentration range.

[0170] Example 2: Selective precipitation of red fluorescent protein with histidine tag using divalent cations

[0171] The crude protein solution 1 (ND) and the crude protein solution 2 (WD) in Example 1 were used. In a similar manner to Example 1, MgCl2 Figure 2a ), MgSO4 Figure 2b ), CaCl2 Figure 2c ), and MnCl2 Figure 2d ) were used, and the fluorescence value in the supernatant was detected by an enzyme marker, and the results are shown in Figures 2a to 2d As can be seen from Figures 2a to 2d , in the concentration range of 1-80 mM, especially in the range of 1.5-40 mM, the ND with a histidine tag is selectively precipitated compared to the non-tagged WD.

[0172] In addition, taking CaCl2 as an example, the yield and purity of the one-step precipitation of ND obtained at different concentrations were investigated. 500 μL of ND was added with 500 μL of CaCl2 solution, so that the corresponding final concentration of Ca 2+ was 5, 10, and 30 mM, respectively, the pH value of the mixed sample was 6.2, and the precipitation was carried out at 4°C for 1 h. Centrifugation was carried out at 12000 rpm for 2 min, the fluorescence and protein concentration in the supernatant were detected, and the one-step precipitation yield and purity of ND were calculated. The purity was calculated in the form of specific fluorescence. The results are shown in Table 2 below.

[0173] Precipitation yield = (original fluorescence - residual fluorescence in supernatant) / original fluorescence

[0174] Specific fluorescence = fluorescence of sample / protein concentration of sample

[0175] Purification fold = specific fluorescence of sample after purification / specific fluorescence of crude protein solution

[0176] Table 2: Yield and purity of one-step precipitation of ND with different concentrations of CaCl2

[0177]

[0178] As Figure 2cAs shown, the WD did not produce precipitates when CaCl2 was added at these concentrations, and as shown in Table 2 above, the target protein ND did not produce precipitates when CaCl2 was added at these concentrations. 2+ The precipitates can be selectively precipitated, and the yield is high, and the purity is greatly improved.

[0179] In addition, similar to the experiment with MgCl2, CoCl2 was added at a concentration of 10 mM 2+ cations or Fe 2+ cations, and it was found that 100% of the ND with a histidine tag was precipitated, while the WD without a histidine tag hardly precipitated at all. In addition, CoCl2 was added at a concentration of 100 mM 2+ cations or Fe 2+ cations, and it was found that all of the ND with a histidine tag was redissolved.

[0180] As a comparative example, similar to the operation with MgCl2, MgCl2 was changed to NiCl2 Figure 2e ), ZnCl2 Figure 2f ), and CuCl2 Figure 2g ), respectively. As can be seen from Figures 2e to 2g , although the target protein ND with a histidine tag was significantly precipitated at an ion concentration of 1 mM, the original protein WD without a tag was also precipitated at most of the concentration ranges, and therefore Ni 2+ , Zn 2+ , Cu 2+ did not have a selective precipitation effect on the target protein.

[0181] From the above results, it can be seen that Ca 2+ , Mg 2+ , Mn 2+ , Co 2+ , Fe 2+ cations can selectively precipitate the red fluorescent protein with a histidine tag in the concentration range of the present application.

[0182] The precipitates of the divalent cations were analyzed. Using the crude protein solution 1 (ND) in Example 1, 2 mM of Ca 2+ , Mg 2+ , Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ was used for the precipitation operation, the system pH was 6.2, and it was left to stand at 4°C for 1 hour, and then centrifuged at 12000 rpm for 2 min, and the results are shown in Figure 3a . In Figure 3a , from left to right are the blank control, Ca2+ Mg 2+ Mn 2+ Co 2+ Ni 2+ Cu 2+ Zn 2+ The precipitation results show that Ni 2+ Cu 2+ Zn 2+ The production of significantly more precipitate indicates that both the target protein and other proteins were precipitated simultaneously. Accordingly, the precipitate and supernatant in each tube were subjected to electrophoresis separately, and the electrophoresis results are as follows: Figure 3b As shown, in Figure 3b The bands from left to right in the middle are the protein standard marker (M), Ca2+, and β2+, respectively. 2+ Supernatant (1), Ca 2+ Precipitate (2), Mg 2+ Supernatant (3), Mg 2+ Precipitate (4), Mn 2+ Supernatant (5), Mn 2+ Precipitation (6), Co 2+ Upper Qing (7), Co 2+ Precipitate (8), Ni 2+ Upper Qing (9), Ni 2+ Precipitate (10), Cu 2+ Supernatant (11), Cu 2+ Precipitate (12), Zn 2+ Shangqing (13), Zn 2+ Precipitation (14). From Figure 3b It can be seen that in Ca 2+ Mg 2+ Mn 2+ Co 2+ The target protein constitutes the majority of the precipitate in the system, while other proteins are mainly retained in the supernatant. In Ni 2+ Cu 2+ Zn 2+ In the system, almost all proteins are in the precipitate, while the supernatant contains very little or almost no protein. Figure 3a and Figure 3b The results show that Ca 2+ Mg 2+ Mn 2+ Co 2+ It has a good selective precipitation effect on the target protein.

[0183] Example 3: Resolubilization of the precipitate and analysis of resolubility selectivity using monovalent cations

[0184] The crude protein solution 1 (ND) from Example 1 was used. Several centrifuge tubes were used, and 500 μL of ND was transferred to each tube. 500 μL of 200 mM NaCl (or NH4Cl) was added to each tube, resulting in a mixed sample pH of approximately 6.2. The mixture was incubated at 4°C for 1 h. After centrifugation, the supernatant and precipitate were separated. The precipitate was resuspended in water at concentrations of 10, 20, 80, 200, 400, and 600 mM NaCl (or NH4Cl). The fluorescence and protein concentration of the resuspended supernatant were measured, and the results are shown in Tables 3 and 4. It can be seen that when the concentration of these monovalent cations is below 30 mM, especially in the 0-20 mM range, or above 300 mM, especially in the 400-600 mM range, the histidine-tagged ND precipitate can be reconstituted, and the purity of the reconstituted protein is improved.

[0185] Reconstitution yield = Fluorescence of supernatant after resuspension / (Fluorescence of original crude protein solution - Residual fluorescence of supernatant after precipitation)

[0186] Purification factor = (fluorescence of resuspended supernatant / protein concentration of resuspended supernatant) / (fluorescence of original crude protein solution / protein concentration of original crude protein solution)

[0187] Table 3: Resuspension of ND precipitates obtained from 100mM NaCl with NaCl solutions of different concentrations

[0188]

[0189] Table 4: Resuspension of ND precipitate obtained with 100mM NH4Cl solutions using NH4Cl solutions of different concentrations

[0190]

[0191] Example 4: Resolubilization of the precipitate and analysis of resolubility selectivity using divalent cations

[0192] Take 950 μl of the crude protein solution 1 (ND) from Example 1, and add 50 μL of a 200 mM MgCl2 aqueous solution to each solution, so that the final Mg content in the system is... 2+ The concentrations were 10 mM, and the pH of the mixed sample was approximately 6.2. The mixture was incubated at 4°C for 1 hour, then centrifuged at 12000 rpm for 2 minutes. The precipitate was separated from the supernatant, and then an aqueous solution of MgCl2 was added to the precipitate to increase the Mg content in the system. 2+ Concentrations of 0 mM, 0.2 mM, 1 mM, 2 mM, 4 mM, 10 mM, 20 mM, 40 mM, 100 mM, and 200 mM were used. After standing at 4℃ for 30 min, the mixture was centrifuged at 12000 rpm for 2 min. The fluorescence absorption of the supernatant at 591 nm under an excitation wavelength of 554 nm was detected using a microplate reader, and the proportion of residual fluorescence in the supernatant was determined. (The text then abruptly shifts to a seemingly unrelated topic about Mg...) 2+The concentration is the abscissa and the ratio of the fluorescence of the original sample is the ordinate. The results are shown in Fig. 2. Figure 4

[0193] As can be seen, when the magnesium ion concentration is lower than 2 mM or higher than 40 mM, the precipitated protein sample is obviously dissolved. Figure 4

[0194] The protein concentration and fluorescence value of the re-dissolved protein solution were detected to evaluate the purification effect of the target protein. It was found that the purity of the target protein in the re-dissolved protein solution was increased by 3, 3.2, 2.7, 4.1 and 4.2 times respectively compared with the original protein solution at the concentrations of 0, 0.2, 1, 40 and 100 mM. This indicates that the target protein can be selectively dissolved during the re-dissolution of the precipitate, thereby increasing the purity of the target protein.

[0195] Example 5: Precipitation and resolubilization of L-threonine aldolase using monovalent cations

[0196] The L-threonine aldolase with a histidine tag (4 subunits, 4 histidine tags) was subjected to the precipitation and re-dissolution operation of monovalent cations.

[0197] 500 μl of L-threonine aldolase crude enzyme solution was taken, and water and 2 M NaCl (or NH4Cl) was added respectively to make the final concentration of cations in the enzyme solution 50, 100 and 200 mM respectively. The pH value of the mixed sample was about 6.2, and it was placed at 4°C for 1 h. After centrifugation, the supernatant and the precipitate were separated, the precipitate sample was resuspended with PB solution (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 8.0), and the enzyme activity and protein concentration of the precipitated sample were detected according to the foregoing method, and the precipitation yield and the purification fold were calculated. The results are shown in Table 5. It can be seen that the L-threonine aldolase with a histidine tag can be precipitated by these monovalent cation concentrations in the range used, and the purity of the precipitated target protein is increased.

[0198] Table 5: Precipitation of L-threonine aldolase using NaCl and NH4Cl

[0199]

[0200] The sample precipitated by 100 mM NH4Cl was re-dissolved with water and 600 mM corresponding monovalent cation solution, and it was placed at 4°C for 1 h. After centrifugation, the supernatant and the precipitate were separated, and the enzyme activity and protein concentration of the re-dissolved supernatant sample were detected according to the foregoing method, and the re-dissolution yield and the purification fold were calculated. The results are shown in Table 6. It can be seen that the L-threonine aldolase with a histidine tag can be re-dissolved by these high-concentration monovalent cation solutions, and the target sample can be dissolved, and the purity of the re-dissolved target protein is increased.

[0201] ​​Table 6: Resuspension of L-threonine aldolase precipitated samples using NH4CI

[0202] Original precipitate Water resuspension supernatant 600 mM NH4 reconstitution supernatant<!-- 14 --> Resolubilization yield (%) 1.00 0.57 0.96 Purification fold 1.00 1.66 3.88

[0203] Example 6: Precipitation and resolubilization of L-threonine aldolase, formate dehydrogenase, L-leucine dehydrogenase using divalent cations Resolubilization

[0204] L-threonine aldolase (4 subunits, 4 histidine tags) was precipitated with different concentrations of divalent cations.

[0205] Take 950 μl of L-threonine aldolase crude enzyme solution, add 50 μl of different concentrations of MgSO4solution, mix well to make the final concentration of magnesium ions 0, 0.1, 0.2, 1, 2, 4, 6, 8, 10, 20, 50, 100 mM, the pH value of the mixed sample is about 6.2, and it is placed at 4°C overnight. Centrifuge at 12000 rpm for 1 min, and take the supernatant and precipitate samples for protein electrophoresis. The results are shown in Figure 5 .

[0206] In Figure 5 , lanes 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 are the supernatant samples after centrifugation after treatment with 0, 0.1, 0.2, 1, 2, 4, 6, 8, 10, 20, 50, 100 mM magnesium ions, lanes 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 are the precipitate samples after centrifugation after treatment with 0.1, 0.2, 1, 2, 4, 6, 8, 10, 20, 50, 100 mM magnesium ions, lane M is the protein marker, and the arrow indicates the target protein band.

[0207] The results show that the target protein does not precipitate under low concentration of magnesium ions, and precipitates obviously in the range of 1-50 mM, but does not precipitate basically at higher concentration (100 mM).

[0208] L-threonine aldolase was precipitated and resuspended with divalent cations, and the electrophoresis results are shown in Figure 6The 950 μl L-threonine aldolase crude enzyme solution (1 lane) was mixed with 50 μl 200 mM MgSO4solution to make the final concentration of magnesium ions 10 mM, and the pH of the mixed sample was about 6.2. After standing for 30 min, the supernatant (2 lane) and protein precipitate (3 lane) were obtained by centrifugal separation. The protein precipitate was resuspended (i.e. redissolved) by blowing with 100 μl 100 mM or 200 mM MgSO4solution, and then centrifugal separation was performed to obtain the precipitate (4 lane, 7 lane). The supernatant containing the target protein was transferred to a new centrifuge tube (the concentration of magnesium ions was 100 or 200 mM). 900 μl deionized water was added to the centrifuge tube containing 100 μl redissolved protein solution to dilute the magnesium ions (the concentration of magnesium ions was changed to 10 or 20 mM). The liquid became turbid, and centrifugal separation was performed to obtain the supernatant (5 lane, 8 lane) and precipitate (6 lane, 9 lane). The precipitate in 6 lane and 9 lane was the purified target protein. 10 lane was Marker, and the arrow indicated the target protein band.

[0209] From Figure 6 It can be seen that the addition of 10 mM magnesium ions to the crude enzyme solution can achieve sufficient precipitation of the target protein. The addition of 100 mM or 200 mM magnesium ions to the precipitate containing 10 mM magnesium ions can make the target protein dissolve well, while the impurities remain in the precipitate. Dilution of the protein solution redissolved with 100 mM or 200 mM magnesium ions can precipitate the target protein sufficiently, and the purity of the target protein is very high.

[0210] The divalent cation precipitation and redissolution operation of formate dehydrogenase (2 subunits, 2 histidine tags) was performed, and the electrophoresis results are shown in Figure 7 The 900 μl formate dehydrogenase crude enzyme solution (1 lane) was mixed with 100 μl 100 mM or 200 mM MgSO4solution to make the final concentration of magnesium ions 10 mM or 20 mM, respectively. The pH of the mixed sample was about 6.2. After standing for 30 min, the supernatant (2 lane and 6 lane) and protein precipitate were obtained by centrifugal separation. The protein precipitate was resuspended (i.e. redissolved) by blowing with 100 μl 200 mM MgSO4solution, and then centrifugal separation was performed to obtain the precipitate (3 lane, 7 lane). The supernatant containing the target protein was transferred to a new centrifuge tube (the concentration of magnesium ions was 200 mM). 900 μl deionized water was added to the centrifuge tube containing 100 μl redissolved protein solution to dilute the magnesium ions (the concentration of magnesium ions was changed to 20 mM). The liquid became turbid, and centrifugal separation was performed to obtain the supernatant (4 lane, 8 lane) and precipitate (5 lane, 9 lane). The precipitate in 5 lane and 9 lane was the purified target protein. M lane was protein marker, and the arrow indicated the target protein band.

[0211] From Figure 7It can be seen that the addition of 10 mM or 20 mM magnesium ions in the crude enzyme solution can achieve the precipitation of the target protein; the addition of 200 mM magnesium ions in the precipitate of 10 mM or 20 mM magnesium ions can make the target protein better dissolved, while a large amount of impurities still remain in the precipitate; dilution of the protein solution resuspended by 200 mM magnesium ions can precipitate the target protein, and the purity of the target protein is higher.

[0212] The precipitation and resuspension operation of divalent cations on L-leucine dehydrogenase containing histidine tag (8 subunits, 8 histidine tags) was carried out, and the electrophoresis results are shown in Figure 8 . Take 900 μl of L-leucine dehydrogenase crude enzyme solution (1 lane), add 100 μl of 100 mM or 200 mM MgSO4solution and mix well, so that the final concentration of magnesium ions is 10 mM or 20 mM respectively, the pH value of the mixed sample is about 6.2, stand for 30 min, centrifugal separation to obtain supernatant (2 lanes and 6 lanes) and protein precipitate; the protein precipitate is resuspended by 100 μl of 200 mM MgSO4solution (i.e. resuspension) and centrifugal separation, to obtain the precipitate (3 lanes, 7 lanes), and the supernatant containing the target protein is moved to a new centrifugal tube (magnesium ion concentration is 200 mM); add 900 μl of deionized water to the centrifugal tube containing 100 μl of resuspended protein solution (so that the concentration of magnesium ions is changed to 20 mM), the liquid becomes turbid, centrifugal, solid-liquid separation, to obtain supernatant (4 lanes, 8 lanes) and precipitate (5 lanes, 9 lanes), and the precipitate of 5 lanes and 9 lanes is the purified target protein. Lane M is protein marker, and the arrow mark is the target protein band.

[0213] From Figure 8 It can be seen that the addition of 10 mM or 20 mM magnesium ions in the crude enzyme solution can achieve the precipitation of the target protein; the addition of 200 mM magnesium ions in the precipitate of 10 mM or 20 mM magnesium ions can make the target protein better dissolved, while a large amount of impurities still remain in the precipitate; dilution of the protein solution resuspended by 200 mM magnesium ions can precipitate the target protein, and the purity of the target protein is higher.

[0214] The protein sample of amino acid ester acyltransferase containing histidine tag (2 subunit proteins, containing 2 histidine tags) was precipitated and resuspended by divalent cations, and the results are shown in Figure 9Add MgSO4 to a final concentration of 10 mM, and the pH of the mixed sample is approximately 6.2. Centrifuge to obtain a supernatant (lane 2) and a precipitate (lane 3). Resuspend and reconstitute the precipitate with MgSO4 to a final concentration of 100 mM, and centrifuge to obtain the precipitate (lane 4). Dilute the 100 mM MgSO4 reconstituted supernatant with water to a MgSO4 concentration of 10 mM. The target protein precipitates again. Centrifuge to obtain a supernatant (lane 5) and the final purified target protein precipitate (lane 6). Lane 1 represents the crude enzyme solution; lane M represents the protein marker; the arrows indicate the target protein band.

[0215] from Figure 9 The results showed that a strategy of low-concentration cation precipitation, high-concentration cation resuspension, and further dilution precipitation could yield target proteins with high yield and high purity.

[0216] Example 7: Effect of system pH on precipitation of histidine-tagged proteins

[0217] Take crude protein solution 1 (ND) from Example 1 and add NaH2PO4 to make the Na content in the system... + The concentration was 100 mmol / L. The pH was adjusted with 100 mM NaOH, and the solution was allowed to stand for 1 hour. The percentage of fluorescent residue in the solution was then measured. The results showed that the precipitation rates of ND were 99%, 99%, 97%, 95%, 21%, and 2% at pH values ​​of 5.0, 5.5, 6.0, 6.6, 7.0, and 7.2, respectively.

[0218] Similarly, NH4Cl was added to crude protein solution 1 (ND) to make the NH4 in the system... + The concentration of ND was 100 mmol / L. The pH was adjusted with 100 mM NH3·H2O. After standing for 1 hour, the percentage of fluorescence residue in the solution was measured. The results showed that the precipitation rates of ND were 99%, 99%, 99%, 97%, 36%, and 2% at pH values ​​of 5.0, 5.5, 6.2, 6.7, 7.1, and 7.3, respectively.

[0219] Similarly, CaCl2 was added to crude protein solution 1 (ND) to increase the Ca content in the system. 2+ The concentration of ND was 10 mmol / L. The pH was adjusted with 10 mM Ca(OH)2. After standing for 1 hour, the percentage of fluorescent residue in the solution was measured. The results showed that the precipitation rate of ND was 100%, 100%, 100%, 88%, 17%, and 3% at pH values ​​of 5.0, 5.5, 6.2, 6.6, 7.0, and 7.2, respectively.

[0220] Similarly, MgCl2 was added to crude protein solution 1 (ND) to increase the Mg content in the system. 2+The concentration of NaOH was 10 mmol / L, and the pH was adjusted with 10 mM NaOH (the Na content in the mixed system was...). + The concentration of the solution was not more than 1 mM. After standing for 1 hour, the percentage of fluorescence residue in the solution was measured. The results showed that the precipitation rate of ND was 100%, 100%, 99%, 97%, 35%, and 7% at pH values ​​of 5.0, 5.5, 6.2, 6.7, 7.0, and 7.2, respectively.

[0221] In addition, 1 mL of the ND bacterial culture from Example 1 was washed with water and centrifuged to obtain bacterial cells. These cells were then resuspended in sodium phosphate solutions with concentrations of 100 mM at pH 5, pH 6, pH 7, and pH 8, respectively. After ultrasonic disruption and centrifugation, the precipitate was resuspended in 1 mL of water. Electrophoresis results are shown below. Figure 10 Lanes 1, 3, 5, and 7 are the supernatants obtained after treatment with pH 8, pH 7, pH 6, and pH 5, respectively; lanes 2, 4, 6, and 8 are the precipitates obtained after treatment with pH 8, pH 7, pH 6, and pH 5, respectively; M is the protein marker; and the arrows indicate the target protein bands.

[0222] The results showed that the target protein ND was almost entirely precipitated at pH 5 and pH 6, a small amount of ND precipitated at pH 7, and no ND precipitated at pH 8. This indicates that a lower pH is necessary for ND precipitation.

[0223] Example 8: Effect of temperature on ND precipitation

[0224] The ND protein sample from Example 1 was taken, and MgCl2 or NaCl was added to a final concentration of 10 mM or 100 mM, respectively. The pH of the mixed sample was approximately 6.2. The samples were then incubated at 4℃, 20℃, and 28℃ for 1 h each. After centrifugation at 12000 rpm for 2 min, the fluorescence and protein concentration of the supernatant were detected. The results are shown in Table 7. The results show that high precipitation yields and purity of the target protein were obtained at different temperatures.

[0225] Table 7: Precipitation results of ND at different temperatures

[0226]

[0227] Example 9: Effect of total protein concentration on precipitation of histidine-tagged target proteins

[0228] The crude protein solution containing the target protein ND from Example 1 was used. Different concentrations of MgCl2 were added to systems with total protein concentrations of 1.16 mg / ml, 0.5 mg / ml, 0.3 mg / ml, 0.17 mg / ml, and 0.05 mg / ml, respectively. The percentage of residual fluorescence in the solution was then measured. See the results below. Figure 11 .from Figure 11 It is evident that the total protein concentration in the solution has no effect on the precipitation effect.

[0229] In a crude protein solution containing 4750 μL of total protein at a concentration of 8.59 mg / mL, 250 μL of 200 mM MgCl2 was added to bring the final concentration to 10 mM MgCl2. The solution was centrifuged at 12000 rpm for 2 min, and the percentage of residual fluorescence was measured. The initial precipitation yield of the target protein ND was calculated to be 98%. The precipitate was resuspended in 5 mL of 100 mM MgCl2, incubated at 4 °C for 1 h, centrifuged at 12000 rpm for 2 min, and the supernatant MgCl2 was diluted to 10 mM with water. After incubation at 4 °C for 1 h, the precipitate was collected after centrifugation at 12000 rpm for 2 min. The yield of the precipitated ND protein was calculated to be 85%, with a purity 3.1 times higher than that of the original crude protein solution.

[0230] Example 10: Cationic selective precipitation of double-histidine-tagged single-subunit proteins

[0231] For the single-subunit green fluorescent protein GFP, recombinant proteins with histidine tags fused to the N-terminus, C-terminus, and both N-terminus and C-terminus were constructed. Crude enzyme solutions containing one or two histidine tags were expressed in *E. coli*. Crude enzyme solutions were prepared for untagged GFP (WG), GFP (CG) with a histidine tag (6 histidine residues) added to the C-terminus, GFP (NG) with a histidine tag (6 histidine residues) added to the N-terminus, and GFP (DG) with six histidine tags (6 histidine residues) added to both ends. 10 mM MgCl2 was added to each crude enzyme solution, and the pH of the mixed sample was approximately 6.2. After centrifugation, the supernatant and precipitate were obtained, and protein electrophoresis was performed on each sample. The results are shown in [Figure number missing]. Figure 12 Lane M represents the protein marker, lane 1 represents the WG crude enzyme solution, lane 2 represents the NG crude enzyme solution, lane 3 represents the CG crude enzyme solution, lane 4 represents the DG crude enzyme solution, lane 5 represents the WG supernatant with added MgCl2, lane 6 represents the NG supernatant with added MgCl2, lane 7 represents the CG supernatant with added MgCl2, lane 8 represents the DG supernatant with added MgCl2, lane 9 represents the WG precipitate with added MgCl2, lane 10 represents the NG precipitate with added MgCl2, lane 11 represents the CG precipitate with added MgCl2, and lane 12 represents the DG precipitate with added MgCl2. The arrows indicate the target protein bands.

[0232] from Figure 12 As can be seen, after the addition of MgCl2, green fluorescent proteins without histidine tags or with only one histidine tag do not precipitate, but proteins with two histidine tags can precipitate.

[0233] For the single subunit protein pyridoxal kinase, the recombinant protein with histidine tag at both N- and C-terminus was constructed, expressed in E. coli and the crude enzyme solution containing the recombinant protein with two histidine tags was prepared. KCl, NaCl and NH4Cl were added in the crude enzyme solution at a final concentration of 100 mM, and the pH value of the mixed sample was about 6.2. The supernatant and precipitate were obtained by centrifugation. The protein electrophoresis results are shown in Figure 13 , in which lane M is a protein marker, lane 1 is the crude enzyme solution, lane 2 is the supernatant with KCl, lane 3 is the precipitate with KCl, lane 4 is the supernatant with NaCl, lane 5 is the precipitate with NaCl, lane 6 is the supernatant with NH4Cl, lane 7 is the precipitate with NH4Cl, and the arrow indicates the target protein band. It can be seen from Figure 13 that the target protein can be selectively precipitated by K + , Na + and NH4 + ions.

[0234] For the single subunit protein pyridoxal kinase, the recombinant protein with histidine tag at both N- and C-terminus was constructed, expressed in E. coli and the crude enzyme solution containing the recombinant protein with two histidine tags was prepared. KCl, NaCl and NH4Cl were added in the crude enzyme solution at a final concentration of 100 mM, and the pH value of the mixed sample was about 6.2. The supernatant and precipitate were obtained by centrifugation. The protein electrophoresis results are shown in Figure 14 , in which lane M is a protein marker, lane 1 is the crude enzyme solution, lane 2 is the supernatant with KCl, lane 3 is the precipitate with KCl, lane 4 is the supernatant with NaCl, lane 5 is the precipitate with NaCl, lane 6 is the supernatant with NH4Cl, lane 7 is the precipitate with NH4Cl, and the arrow indicates the target protein band. It can be seen from Figure 14 that the target protein can be selectively precipitated by K + , Na + and NH4 + ions.

[0235] Example 11: Purification protocol of precipitating impurities at high cation concentration and then diluting the target protein precipitate

[0236] In 500 μL of the crude protein solution of red fluorescent protein ND with histidine tag in Example 1, 500 μL of 200 mM MgCl2 was added to make the final concentration of MgCl2 100 mM, and the pH value of the mixed sample was about 6.2. It was placed at 4°C for 1 h. It was centrifuged at 12000 rpm for 2 min. The obtained supernatant was diluted with water to make the final concentration of MgCl2 10 mM, and it was placed at 4°C for 1 h. It was centrifuged at 12000 rpm for 2 min, and the target protein yield in the precipitate was 96%, which was 3.68 times higher than the purity of the crude protein solution.

[0237] Similarly, the ND crude protein solution was added with NaCl to a final concentration of 600 mM, the pH of the mixed sample was about 6.2, and the sample was left to stand at 4°C for 1 h. The sample was centrifuged at 12 000 rpm for 2 min, and the obtained supernatant was diluted with water to a final concentration of 100 mM NaCl, and the sample was left to stand at 4°C for 1 h. The sample was centrifuged at 12 000 rpm for 2 min, and the obtained precipitate was collected. The yield of the target protein in the precipitate was 96%, and the purity was increased by 4.08 times compared with the crude protein solution.

[0238] This shows that part of the impurity proteins can be precipitated by using a solution of a higher concentration of monovalent or divalent cations, and the target protein with a histidine tag remains in the supernatant. The supernatant obtained by centrifugation is diluted with water to a low concentration, and the impurity proteins remain in the supernatant, while the target protein with a histidine tag is precipitated, thereby achieving the purification of the target protein.

[0239] Example 12: Purification protocol of precipitating at low cation concentration, resuspending at high cation concentration, and then diluting the precipitate

[0240] The E. coli PET28a plasmid was used as a vector to clone and express L- leucine dehydrogenase (LDH, 8 subunits, 8 tags) with 6 histidine tags at the N terminus. The obtained bacterial cells were resuspended with deionized water, and the crude protein solution was obtained after the bacterial cells were broken. The purification method for the monovalent cations sodium and ammonium was as follows: 50 μL of 2 M NaCl or NH4Cl (final concentration of 100 mM NaCl or NH4Cl) was added to 950 μL of the crude protein solution, the pH of the mixed sample was about 6.2, the sample was left to stand at 4°C for 1 h, and then centrifuged at 12 000 rpm for 2 min. The precipitate was resuspended and dissolved with 100 μL of 800 mM NaCl, left to stand for 1 h, and then the resuspended solution was centrifuged at 12 000 rpm for 2 min. Deionized water was added to the supernatant after centrifugation to dilute the NaCl or NH4Cl to a concentration of 80 mM, and then centrifuged at 12 000 rpm for 2 min. The obtained precipitate was the high-purity target protein LDH.

[0241] The purification method for the divalent cation magnesium was as follows: 50 μL of 200 mM MgSO4 (final concentration of 10 mM MgSO4) was added to 950 μL of the crude protein solution, the pH of the mixed sample was about 6.2, the sample was left to stand at 4°C for 1 h, and then centrifuged at 12 000 rpm for 2 min. The precipitate was resuspended and dissolved with 100 μL of 100 mM MgSO4 / CaCl2, left to stand for 1 h, and then the resuspended solution was centrifuged at 12 000 rpm for 2 min. Deionized water was added to the supernatant after centrifugation to dilute the MgSO4 to a concentration of 10 mM, and then centrifuged at 12 000 rpm for 2 min. The obtained precipitate was the high-purity target protein LDH.

[0242] Metal ion affinity chromatography (IMAC) was used as a comparison. GE's HiTrap prepacked column (Ni-IMAC) was used to purify LDH. First, the column was washed with five column volumes of deionized water at 3 mL / min; the enzyme solution to be purified was mixed with binding buffer (20 mM PB, 500 mM NaCl, 20 mM imidazole, pH 8.0) at 1:1, and then loaded at 1 mL / min; the unbound proteins were washed with five column volumes of binding buffer; and the target protein was eluted with five column volumes of elution buffer (20 mM PB, 500 mM NaCl, 500 mM imidazole, pH 8.0).

[0243] The samples precipitated with sodium, ammonium, and magnesium ions and the IMAC purified sample were subjected to protein electrophoresis, and the results are shown in Figure 15 wherein lane M is a protein marker, lane 1 is a crude enzyme solution, lane 2 is the IMAC purified LDH, lane 3 is the magnesium ion purified sample, lane 4 is the sodium ion purified sample, lane 5 is the ammonium ion purified sample, and the arrow indicates the target protein band.

[0244] From Figure 15 it can be seen that the target protein obtained by the magnesium, sodium, and ammonium precipitation methods has a purity comparable to that of the IMAC purified sample, indicating that the method of the present application has a very good purification effect on L-leucine dehydrogenase containing a histidine tag.

[0245] The same method was used for red fluorescent protein (ND, 4 subunits, 4 tags) with a histidine tag (6 histidine residues) at the N-terminus and L-threonine aldolase (LTA, 4 subunits, 4 tags) with a histidine tag, and the results are shown in Figure 16 and Figure 17 In Figure 16 , lane M is a protein marker, lane 1 is the IMAC purified ND, lane 2 is a crude enzyme solution, lane 3 is the magnesium ion purified sample, lane 4 is the sodium ion purified sample, lane 5 is the ammonium ion purified sample, and the arrow indicates the target protein band. In Figure 17 , lane M is a protein marker, lane 1 is a crude enzyme solution, lane 2 is the IMAC purified LTA, lane 3 is the magnesium ion purified sample, lane 4 is the sodium ion purified sample, lane 5 is the ammonium ion purified sample, and the arrow indicates the target protein band.

[0246] From Figure 16 and Figure 17 it can be seen that the method of the present application also has a very good purification effect on red fluorescent protein and L-threonine aldolase containing a histidine tag.

[0247] Example 13: Effect of different lengths of histidine tag on precipitation

[0248] Fluorescent proteins with 3 histidine residues, 9 histidine residues and 12 histidine residues (3ND, 9ND, 12ND) were constructed respectively, and crude protein solutions were obtained. 500 μl of the crude protein solution was added with different volumes of 2 mol / l MgCl2 aqueous solution and deionized water to make the total volume of the system 1 ml, and the Mg 2+ concentration was 3 mM and 8 mM respectively, the pH value of the mixed sample was about 6.2, and the sample was placed at 4°C for 1 hour, and then centrifuged at 12000 rpm for 2 min. The fluorescence absorption of the supernatant at 554 nm excitation wavelength and 591 nm was detected by an enzyme marker, and the residual fluorescence percentage in the solution was detected. It was found that under the condition of 3 mM Mg 2+ , the precipitation rates of 3ND, 9ND and 12ND were 93.2%, 98.2% and 93% respectively; and under the condition of 8 mM Mg 2+ , the precipitation rates of 3ND, 9ND and 12ND were 82.5%, 99.7% and 99.4% respectively.

[0249] Example 14: Precipitation-dissolution of proteins using different combinations of ions

[0250] For the ND protein sample in Example 1, MgSO4 was added to a final concentration of 10 mM for precipitation, and the obtained precipitate was resuspended with 100 mM CaCl2 or 500 mM NH4Cl, the pH value of the mixed sample was about 6.2, and the supernatant was obtained by centrifugation. The sample was diluted with water (calcium was diluted to 10 mM, and ammonia was diluted to 100 mM), and electrophoresis was performed on each sample. The results are shown in Figure 18 , wherein lane 1 is the original ND sample; lane 2 is the supernatant obtained by centrifugation after treatment with 10 mM MgSO4; lane 3 is the residual precipitate after resuspension of the precipitate with 100 mM CaCl2; lane 4 is the precipitate obtained by centrifugation after dilution of the supernatant obtained by resuspension of the precipitate with 100 mM CaCl2 with water; lane 5 is the supernatant obtained by centrifugation after dilution of the supernatant obtained by resuspension of the precipitate with 100 mM CaCl2 with water; lane 6 is the residual precipitate after resuspension of the precipitate with 500 mM NH4Cl; lane 7 is the precipitate obtained by centrifugation after dilution of the supernatant obtained by resuspension of the precipitate with 500 mM NH4Cl with water; lane 8 is the supernatant obtained by centrifugation after dilution of the supernatant obtained by resuspension of the precipitate with 500 mM NH4Cl with water; lane M is a protein marker; and the arrow indicates the target protein band.

[0251] Figure 18 The results show that the target protein can be obtained with high yield and high purity by precipitating and dissolving the ND sample with different combinations of ions.

Claims

1. A method for isolating and purifying a protein bearing at least two histidine tags, the method comprising: contacting said protein in a solubilized state with cations in an aqueous solution, to obtain a precipitate comprising said protein; wherein the pH of the aqueous solution is in the range of less than or equal to 7.2; The cations are selected from one or more of the following: non-H + monovalent cations, Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ ; when the cation is selected from monovalent cations other than H + 10-400 mmol / L; when the cation is selected from Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ and Co 2+ , the concentration of the cation in the aqueous solution is 1-80 mmol / L.

2. The method of claim 1, wherein the non-H + monovalent cation is selected from one or more of NH4 + , Na + , K + , and organic nitrogen-containing cations.

3. The method of claim 2, wherein the organic nitrogen-containing cations are derived from small organic molecules containing -NH2, -NH- and / or =N- groups.

4. The method of claim 3, wherein the organic nitrogen-containing cations are derived from imidazole, glucosamine, guanidine hydrochloride, lysine, arginine and histidine.

5. The method of claim 1, wherein the histidine tags are located at the N-terminus and / or C-terminus of the protein subunits.

6. The method of claim 5, wherein the histidine tags are short peptides consisting of a string of 2-15 consecutive histidine residues.

7. The method of claim 6, wherein the histidine tags are short peptides consisting of 3, 6, 9 or 12 histidine residues.

8. The method of any one of claims 1-7, wherein the pH of the aqueous solution is in the range of 5.0-7.

0.

9. The method of any one of claims 1-7, wherein the pH of the aqueous solution is in the range of 6.0-7.

0.

10. The method of any one of claims 1-7, wherein when the cation is selected from the group consisting of non-H + monovalent cations, the concentration of the cation in the aqueous solution is from 15 to 300 mmol / L.

11. The method of any one of claims 1-7, wherein when the cation is selected from the group consisting of non-H + monovalent cations, the concentration of the cation in the aqueous solution is 30-200 mmol / L.

12. The method of any one of claims 1-7, wherein when the cation is selected from the group consisting of Mg 2+ ,Ca 2+ ,Mn 2+ ,Fe 2+ and Co 2+ , the concentration of the cation in the aqueous solution is 1.5-40 mmol / L.

13. The method of any one of claims 1-7, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ , the concentration of the cation in the aqueous solution is 3-30 mmol / L.

14. A method for isolating and purifying a protein bearing at least two histidine tags, said method comprising: a) contacting said protein in a solubilized state with cations at a first concentration in an aqueous solution, to obtain a precipitate comprising said protein; b) separating said precipitate from the supernatant; c) contacting the separated precipitate with cations at a second concentration in an aqueous solution, to cause at least a portion of said protein contained in said precipitate to resolubilize; wherein the pH of the aqueous solution in steps a) and c) is independently in the range of less than or equal to 7.2; the cations in step a) and step c) are each independently selected from one or more of the following: non-H + monovalent cations, Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ ; In step a), when the cations are selected from non-H + monovalent cations, the first concentration is 15-300 mmol / L; when the cations are selected from Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ and Co 2+ , the first concentration is 1.5-40 mmol / L; In step c), when the cation is selected from non-H + monovalent cations other than H+, the second concentration is greater than or equal to 250 mmol / L but not more than 1000 mmol / L, or the second concentration is less than or equal to 30 mmol / L. In step c), when the cation is selected from Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ and Co 2+ , the second concentration is greater than or equal to 35 mmol / L but not more than 250 mmol / L, or the second concentration is less than or equal to 2 mmol / L.

15. The method of claim 14, further comprising step d) separating the supernatant obtained in step c) and adjusting the cation concentration therein to be within the range of said first concentration, to cause at least a portion of said protein in the supernatant to precipitate again.

16. The method of claim 14, wherein the non-H + monovalent cation is selected from one or more of NH4 + , Na + , K + , and organic nitrogen-containing cations.

17. The method of claim 16, wherein the organic nitrogen-containing cations are derived from small organic molecules containing -NH2, -NH- and / or =N- groups.

18. The method of claim 17, wherein the organic nitrogen-containing cations are derived from imidazole, glucosamine, guanidine hydrochloride, lysine, arginine and histidine.

19. The method of claim 14, wherein the histidine tags are located at the N-terminus and / or C-terminus of the protein subunits.

20. The method of claim 19, wherein the histidine tags are short peptides consisting of a string of 2-15 consecutive histidine residues.

21. The method of claim 20, wherein the histidine tags are short peptides consisting of 3, 6, 9 or 12 histidine residues.

22. The method of any one of claims 14-21, wherein the pH of the aqueous solution in steps a) and c) is independently in the range of 5.0-7.

0.

23. The method of any one of claims 14-21, wherein the pH of the aqueous solution in step a) and step c) is independently in the range of 6.0-7.

0.

24. The method of any one of claims 14-21, wherein the first concentration is 30-200 mmol / L when the cation is selected from the group consisting of non-H + monovalent cations other than H.

25. The method of any one of claims 14-21, wherein the first concentration is 80-120 mmol / L when the cation is selected from the group consisting of non-H + monovalent cations other than H.

26. The method of any one of claims 14-21, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ , the first concentration is 3-30 mmol / L.

27. The method of any one of claims 14-21, wherein when the cation is selected from the group consisting of non-H + monovalent cations, the second concentration is greater than or equal to 400 mmol / L.

28. The method of any one of claims 14-21, wherein when the cation is selected from the group consisting of non-H + monovalent cations, the second concentration is greater than or equal to 600 mmol / L.

29. The method of any one of claims 14-21, wherein when the cation is selected from the group consisting of non-H + monovalent cations, the second concentration is no more than 800 mmol / L.

30. The method of any one of claims 14-21, wherein when the cation is selected from the group consisting of non-H + monovalent cations, the second concentration is less than or equal to 20 mmol / L.

31. The method of any one of claims 14-21, wherein when the cation is selected from the group consisting of non-H + monovalent cations, the second concentration is less than or equal to 10 mmol / L.

32. The method of any one of claims 14-21, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ , the second concentration is greater than or equal to 50 mmol / L.

33. The method of any one of claims 14-21, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ , the second concentration is greater than or equal to 100 mmol / L.

34. The method of any one of claims 14-21, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ , the second concentration is no more than 200 mmol / L.

35. The method of any one of claims 14-21, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ , the second concentration is less than or equal to 1 mmol / L.

36. A method for isolating and purifying a protein bearing at least two histidine tags, the method comprising: a) contacting the protein in a solubilized state with a mixture of impurities and a third concentration of a cation in an aqueous solution, to obtain a precipitate comprising the impurities and a supernatant comprising the protein; b) separating the precipitate from the supernatant; and c) diluting the separated supernatant to a fourth concentration of a cation, such that at least a portion of the protein contained in the supernatant precipitates; wherein the pH of the aqueous solution in step a) and step c) is independently in the range of less than or equal to 7.2; the cations in step a) and step c) are each independently selected from one or more of the following: non-H + monovalent cations, Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ ; In step a), when the cations are selected from non-H + monovalent cations, the third concentration is greater than or equal to 250 mmol / L, but does not exceed 1000 mmol / L; when the cations are selected from Mg 2+ , Ca 2+ , Fe 2+ , Mn 2+ and Co 2+ , the third concentration is greater than or equal to 35 mmol / L, but does not exceed 250 mmol / L; In step c) when the cations are selected from non-H + monovalent cations, the fourth concentration is comprised between 15 and 300 mmol / L; when the cations are selected from Mg 2+ , Ca 2+ , Fe 2+ , Mn 2+ and Co 2+ , the fourth concentration is comprised between 1.5 and 40 mmol / L.

37. The method of claim 36, further comprising step d): separating the precipitate obtained in step c) and contacting it with a second concentration of a cation in an aqueous solution, such that at least a portion of the protein contained in the precipitate redissolves; when the cation is selected from non-H + monovalent cations, the second concentration is greater than or equal to 250 mmol / L but no more than 1000 mmol / L, or the second concentration is less than or equal to 30 mmol / L; when the cation is selected from Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ , the second concentration is greater than or equal to 35 mmol / L but no more than 250 mmol / L, or the second concentration is less than or equal to 2 mmol / L.

38. The method of claim 36, wherein, said non-H + monovalent cation is selected from one or more of NH4 + , Na + , K + and organic nitrogen-containing cations.

39. The method of claim 38, wherein, the organic nitrogen-containing cation is derived from an organic small molecule containing -NH2, -NH- and / or =N- groups.

40. The method of claim 39, wherein, the organic nitrogen-containing cation is derived from imidazole, glucosamine, guanidine hydrochloride, lysine, arginine and histidine.

41. The method of claim 36, wherein, the histidine tag is located at the N-terminus and / or C-terminus of a protein subunit.

42. The method of claim 41, wherein, the histidine tag is a short peptide consisting of a string of 2-15 consecutive histidine residues.

43. The method of claim 42, wherein, the histidine tag is a short peptide consisting of 3, 6, 9 or 12 histidines.

44. The method of any one of claims 36-43, wherein the pH of the aqueous solution in step a) and step c) is independently in the range of 5.0-7.

0.

45. The method of any one of claims 36-43, wherein the pH of the aqueous solution in step a) and step c) is independently in the range of 6.0-7.

0.

46. The method of any one of claims 36-43, wherein the third concentration is greater than or equal to 400 mmol / L when the cation is selected from the group consisting of non-H + monovalent cations.

47. The method of any one of claims 36-43, wherein the third concentration is greater than or equal to 600 mmol / L when the cation is selected from the group consisting of non-H + monovalent cations other than H.

48. The method of any one of claims 36-43, wherein when the cation is selected from the group consisting of non-H + monovalent cations, the third concentration is no more than 800 mmol / L.

49. The method of any one of claims 36-43, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Fe 2+ , Mn 2+ , and Co 2+ , the third concentration is greater than or equal to 50 mmol / L.

50. The method of any one of claims 36-43, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Fe 2+ , Mn 2+ , and Co 2+ , the third concentration is greater than or equal to 100 mmol / L.

51. The method of any one of claims 36-43, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Fe 2+ , Mn 2+ , and Co 2+ , the third concentration is no more than 200 mmol / L.

52. The method of any one of claims 36-43, wherein when the cation is selected from the group consisting of non-H + monovalent cations, the fourth concentration is 30-200 mmol / L.

53. The method of any one of claims 36-43, wherein the fourth concentration is 80-120 mmol / L when the cation is selected from the group consisting of non-H + monovalent cations other than H.

54. The method of any one of claims 36-43, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Fe 2+ , Mn 2+ , and Co 2+ , the fourth concentration is 3-30 mmol / L.

55. The method of claim 37, wherein when the cation is selected from the group consisting of non-H + monovalent cations, the second concentration is greater than or equal to 400 mmol / L.

56. The method of claim 37, wherein when the cation is selected from the group consisting of non-H + monovalent cations other than Na+, the second concentration is greater than or equal to 600 mmol / L.

57. The method of claim 37, wherein when the cation is selected from the group consisting of non-H + monovalent cations, the second concentration is no more than 800 mmol / L.

58. The method of claim 37, wherein when the cation is selected from the group consisting of non-H + monovalent cations, the second concentration is less than or equal to 20 mmol / L.

59. The method of any one of claims 37-43, wherein when the cation is selected from the group consisting of non-H + monovalent cations, the second concentration is less than or equal to 10 mmol / L.

60. The method of any one of claims 37-43, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ , the second concentration is greater than 50 mmol / L.

61. The method of any one of claims 37-43, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ , the second concentration is greater than or equal to 100 mmol / L.

62. The method of any one of claims 37-43, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ , the second concentration is no more than 200 mmol / L.

63. The method of any one of claims 37-43, wherein when the cation is selected from the group consisting of Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , and Co 2+ , the second concentration is less than or equal to 1 mmol / L.

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