Protein stabilizer

Abstract

To provide a protein stabilizer capable of stabilizing a protein in a solution state over a long period without affecting a measurement system.SOLUTION: A protein stabilizer comprises, as an active ingredient, a compound represented by formula (1). [In formula (1), n is an integer from 5 to 15.]SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a protein stabilizer for use with proteins in solution.

Background Art

[0002] Currently, analysis of target substances is widely carried out in various fields such as clinical diagnosis, food hygiene, and environmental hygiene. For example, in the field of clinical diagnosis, analysis of endogenous substances such as enzymes and lipids and exogenous substances such as viruses is performed, and in the field of food hygiene, analysis of residual proteins and allergenic substances in the production environment is carried out. As measurement methods commonly used in inspections, measurement methods using enzyme reactions, immune reactions, and antigen-antibody reactions can be mentioned. In these measurement systems, proteins such as enzymes, antigens, and antibodies are used as components of the reagent, and it is necessary to always obtain the same result even when measuring repeatedly. For this reason, it is required to stabilize these proteins. Many proteins are easily denatured and inactivated by various factors such as temperature, light, pH, salt concentration, and oxidation, and lose their physiological activity. Therefore, when storing proteins, it is important to protect the proteins from these external factors and maintain their physiological activity. In particular, it is known that the stability of proteins is likely to decrease due to the influence of storage temperature.

[0003] Therefore, as a method for stabilizing these proteins, a method of improving stability by freeze-drying is known (see, for example, Non-Patent Document 1). By making the protein into a dried body, chemical changes and structural changes due to molecular movement in an aqueous solution can be suppressed. In addition, although proteins have the property of being easily destabilized by heat, the freeze-drying method can prepare a protein freeze-dried body without applying heat. In addition, stabilization of proteins by suppressing aggregation of proteins using a water-soluble polymer compound such as polyethylene glycol has also been reported (see, for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Document

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] However, in the method for stabilizing proteins by freeze-drying described in Non-Patent Document 1, there is a risk of denaturing the proteins due to the dehydration operation or the dissolution operation when using the proteins. Further, in the stabilization method of adding the polymer compound described in Patent Document 1, there is a problem that when the antigen-antibody reaction is carried out in a state where the polymer compound is present at a high concentration, reaction inhibition is caused. In view of the above problems, an object of the present invention is to provide a protein stabilizer that does not affect the measurement system and can stably maintain proteins in a solution state for a long period even under room temperature conditions.

Means for Solving the Problems

[0007] As a result of intensive studies in view of the above problems, the present inventors have found that the compound represented by the following formula (1) stabilizes proteins in a solution containing water, and have completed the present invention.

[0008] That is, the present invention is as follows [1] to [4]. [1] A protein stabilizer containing, as an active ingredient, a compound represented by formula (1).

Chemical Formula

[0009] The compound of the formula (1), which is the active ingredient of the protein stabilizer of the present invention, can easily retain the activity of the protein and achieve long-term stabilization only by co-existing with the protein in a solution containing water. Further, a protein stabilizing solution obtained by mixing the protein stabilizer of the present invention with a protein (for example, a plasma preparation protein, a labeled immunologically active substance or an enzyme) can be stably stored for a long time, and can be widely used in the fields of clinical tests and diagnostic agents in measuring methods using immunological reactions such as enzyme immunoassay and chemiluminescent enzyme immunoassay. [Modes for Carrying Out the Invention]

[0010] The present invention will be described in detail below. In the following description, a protein solution means a solution in which only a protein is dissolved, a protein stabilizing solution means a solution in which both a protein and the protein stabilizer of the present invention are dissolved, and a protein stabilizer solution means the protein stabilizer of the present invention in a solution form containing the compound of the formula (1) and water without containing a protein. The active ingredient of the protein stabilizer of the present invention is a compound represented by the formula (1). [Chemical formula] In the formula (1), n is an integer of 5 to 15. From the perspective of enhancing the effect of long-term protein stabilization, the preferred range of n is as follows. When the protein is peroxidase and the storage temperature is 0 to 10°C, preferably 2 to 6°C, n is preferably 5 to 15, more preferably 5 to 7. When the storage temperature is 10 to 40°C, preferably 20 to 30°C, n is preferably 7 to 15, more preferably 13 to 15. When the protein is alkaline phosphatase and the storage temperature is 10 to 40°C, preferably 20 to 30°C, n is preferably 5 to 13, more preferably 5 to 7. When the storage temperature is 20 to 50°C, preferably 30 to 40°C, n is preferably 5 to 9, more preferably 5 to 7.

[0011] The compound of formula (1) can be synthesized, for example, by reacting 2-chloro-2-oxo-1,3,2-dioxaphospholane with 1-alkanol in the presence of an amine catalyst such as diisopropylamine in acetonitrile, and then adding trimethylamine (TMA) to the obtained compound and reacting at room temperature to 80°C for 10 to 50 hours. As the 1-alkanol, 1-alkanol having 6 to 16 carbon atoms is preferred.

[0012] The protein stabilizer of the present invention preferably contains water. As the water, purified water, pure water, ion-exchanged water, etc. are preferred. Further, the protein stabilizer of the present invention may be a solution in which the compound represented by formula (1) is dissolved in various buffer solutions containing water. As the various buffer solutions, buffer solutions usually used in this field can be used as long as they do not lose the physiological activities such as the enzyme activity and antigenicity of the protein. For example, phosphate buffer, Tris buffer, Good buffer, glycine buffer, borate buffer, etc. can be mentioned, and these may be mixed and used. A solution containing the compound represented by formula (1) and water or various buffer solutions may be referred to as a protein stabilizer solution as described above.

[0013] The compound represented by the formula (1) contained in the protein stabilizer solution is preferably 0.001% by mass or more, more preferably 0.01% by mass or more. The upper limit is not particularly limited as long as it is soluble in water which is the main solvent. For example, it is 20% by mass or less, preferably 10% by mass or less. Within these ranges, the protein stabilizer solution exhibits an effective protein stabilizing effect and can preferably dissolve the protein or mix well with the protein solution.

[0014] Specific examples of the compound represented by the formula (1) include hexyl(2-[trimethylammonio]ethyl)phosphate (n = 5 in the formula (1)), octyl(2-[trimethylammonio]ethyl)phosphate (n = 7 in the formula (1)), decyl(2-[trimethylammonio]ethyl)phosphate (n = 9 in the formula (1)), dodecyl(2-[trimethylammonio]ethyl)phosphate (n = 11 in the formula (1)), tetradecyl(2-[trimethylammonio]ethyl)phosphate (n = 13 in the formula (1)), hexadecyl(2-[trimethylammonio]ethyl)phosphate (n = 15 in the formula (1)), and the like.

[0015] In addition to the compound represented by the formula (1), compounds that can be contained in the protein stabilizer include other reagents and the like that are usually used in this field for the purpose of further stabilizing the protein. Examples include saccharides, proteins other than the protein to be stabilized, salts, surfactants, and the like. Examples of saccharides include lactose, sucrose, trehalose, and the like. Examples of proteins other than the protein to be stabilized include bovine serum albumin, gelatin, casein, and the like. Examples of salts include amino acids and amino acid salts such as glycine, alanine, serine, threonine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, histidine, etc., peptides such as glycylglycine, inorganic salts such as phosphates, borates, sulfates, tris salts, etc., flavins, organic acids such as acetic acid, citric acid, malic acid, maleic acid, gluconic acid, and salts of organic acids. Examples of surfactants include polyoxyethylene alkyl ethers and the like.

[0016] In the present invention, a method for stabilizing a protein can be provided, which includes co-existing the protein and the compound represented by formula (1) in a solution containing water. By this method, the protein can be stabilized in water for a long period of time. The method of using the protein stabilizer of the present invention will be described below.

[0017] The protein stabilized by the protein stabilizer of the present invention is not particularly limited, and examples include enzymes such as acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, glucoamylase, glucose oxidase, glucose-6-phosphate dehydrogenase, hexokinase, penicillinase, peroxidase, lysozyme, DNA polymerase, albumins, blood coagulation factors, plasma proteins such as immunoglobulins and C-reactive protein (CRP), rheumatoid factor (RF), antibodies against the above proteins, and proteins prepared by genetic recombination, etc. Preferably, peroxidase, alkaline phosphatase, etc. widely used in enzyme immunoassay are included. The protein stabilized by the protein stabilizer may be a single one or may contain two or more kinds. Such a protein may be dissolved in a solvent to form a protein solution before being stabilized by the use of the protein stabilizer of the present invention. As such a solvent, a buffer solution commonly used in this field can be used as long as it does not cause loss of physiological activities such as enzyme activity and antigenicity of the protein. For example, phosphate buffer, Tris buffer, Good buffer, glycine buffer, borate buffer, etc. can be mentioned. Also, these may be mixed and used.

[0018] The protein stabilizer of the present invention can be used for stabilizing the contained protein in a measurement system using, for example, an antibody labeled with such a protein. The protein stabilizer of the present invention can be added to a protein solution for use. Alternatively, the protein stabilizer of the present invention may be used as a protein stabilizer solution to dissolve the target protein. Furthermore, a protein solution and a protein stabilizer solution may be prepared and the two solutions may be mixed. In any case, the concentration of the compound of formula (1) in the protein stabilization solution in which the protein and the protein stabilizer coexist is preferably 0.001 to 10% by mass, more preferably 0.01 to 10% by mass. If it is less than 0.001% by mass, the protein stabilization effect is not sufficient, and if it exceeds 10% by mass, the solution is likely to foam and may be difficult to use. When stabilizing a protein with the protein stabilizer of the present invention, the temperature at which the protein stabilization solution is held is preferably 0°C to 60°C, more preferably 0°C to 50°C.

[0019] Furthermore, the protein stabilization solution may contain saccharides such as glucose, fructose, and cyclodextrin, surfactants such as polyoxyethylene alkyl ether and polyethylene glycol alkyl ether, and proteins such as bovine serum albumin, which are commonly used in this field for the purpose of further stabilizing the protein. Other compounds include those described above that can be contained in the protein stabilizer of the present invention.

Examples

[0020] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited thereto. In this example, the compound of formula (1) shown in each of the following synthesis examples was used as the active ingredient of each protein stabilizer.

[0021] <Synthesis of the compound shown in formula (1)> (Synthesis Example 1) 17.11 g (0.12 mol) of 2-chloro-2-oxo-1,3,2-dioxaphospholane and 12.2 g (0.12 mol) of 1-hexanol were dissolved in 100 g of acetonitrile (MeCN). 13.37 g (0.13 mol) of diisopropylamine was added as a base, and the mixture was reacted at 0 °C for 2 hours. Then, the reaction solution was filtered and purified. Next, 93.67 g (0.24 mol) of trimethylamine (TMA) was added to the obtained compound, and the mixture was reacted at 75 °C for 14 hours. After desolvation and purification by recrystallization, 9.6 g (yield: 30%) of a white powder of hexyl(2-[trimethylammonio]ethyl)phosphate (n = 5), which is the compound represented by formula (1), was obtained.

[0022] (Synthesis Example 2) Using 1-octanol instead of 1-hexanol and changing the charged amounts so that the molar ratio was the same as in Synthesis Example 1, 12.3 g (yield: 35%) of a white powder of octyl(2-[trimethylammonio]ethyl)phosphate (n = 7 in formula (1)), which is the compound represented by formula (1), was obtained in the same manner as in Synthesis Example 1.

[0023] (Synthesis Example 3) Using 1-decanol instead of 1-hexanol and changing the charged amounts so that the molar ratio was the same as in Synthesis Example 1, 12.4 g (yield: 32%) of a white powder of decyl(2-[trimethylammonio]ethyl)phosphate (n = 9 in formula (1)), which is the compound represented by formula (1), was obtained in the same manner as in Synthesis Example 1.

[0024] (Synthesis Example 4) Using lauryl alcohol instead of 1-hexanol and changing the charged amounts so that the molar ratio was the same as in Synthesis Example 1, 12.2 g (yield: 29%) of a white powder of dodecyl(2-[trimethylammonio]ethyl)phosphate (n = 11 in formula (1)), which is the compound represented by formula (1), was obtained in the same manner as in Synthesis Example 1.

[0025] (Synthesis Example 5) Using 1-tetradecanol instead of 1-hexanol and changing the charged amount so that the molar ratio was the same as in Synthesis Example 1, 16.4 g (yield: 36%) of a white powder of tetradecyl(2-[trimethylammonio]ethyl)phosphate (n = 13 in formula (1)), which is a compound represented by formula (1), was obtained in the same manner as in Synthesis Example 1.

[0026] (Synthesis Example 6) Using 1-hexadecanol instead of 1-hexanol and changing the charged amount so that the molar ratio was the same as in Synthesis Example 1, 19.6 g (yield: 40%) of a white powder of hexadecyl(2-[trimethylammonio]ethyl)phosphate (n = 15 in formula (1)), which is a compound represented by formula (1), was obtained in the same manner as in Synthesis Example 1.

[0027] <Example 1-1: Examples 1-1-1 to 1-1-4> <Preparation of Protein Stabilizer Solution> The compound obtained in Synthesis Example 1 was dissolved in Dulbecco’s Phosphate Buffered Saline (manufactured by Sigma-Aldrich, hereinafter abbreviated as phosphate buffer solution (pH = 7.4)) to prepare a protein stabilizer solution.

[0028] <Evaluation of Protein Stabilization Effect> (1) The prepared protein stabilizer solution was mixed with a phosphate buffer solution containing peroxidase-labeled anti-mouse IgG goat antibody (manufactured by Bio-Rad) and sucrose (manufactured by Fujifilm Wako Pure Chemical Corporation) to prepare a protein stabilization solution. The concentration of peroxidase-labeled anti-mouse IgG goat antibody in the protein solution was 0.01% by mass, and the sucrose concentration was 10% by mass. (2) It was incubated at the number of days described in Table 1, 4 °C and 25 °C. After incubation, 4 μL / well of the protein stabilization solution was added to a 96-well polystyrene plate, and 100 μL / well of a color development solution prepared by mixing Peroxidase Substrate Solution B (manufactured by LGC Clinical Diagnostics, Inc (SeraCare)) and TMB Peroxidase Substrate (manufactured by LGC Clinical Diagnostics, Inc (SeraCare)) at a ratio of 9 to 1 was added, and the color development reaction with the protein was carried out for 3 minutes. Subsequently, 50 μL / well of 2N sulfuric acid (manufactured by Fujifilm Wako Pure Chemical Corporation) was added to stop the color development reaction. The absorbance at a wavelength of 450 nm was measured, and the stabilization effect of the peroxidase-labeled anti-mouse IgG goat antibody was evaluated. That is, the absorbance immediately after the preparation of the protein stabilization solution and the absorbance after the passage of time by the above incubation were measured by the following method, and the residual enzyme activity rate (%) was calculated by the following formula (1). The protein stabilization effect was evaluated by the residual enzyme activity rate (%), and the higher the value of the residual enzyme activity rate, the higher the protein stabilization effect. The evaluation results under the storage condition of 4 °C are shown in Tables 1 and 2, and the evaluation results under the storage condition of 25 °C are shown in Tables 3 and 4.

[0029] <Absorbance measurement method> The absorbance of each protein stabilization solution was measured under the following measurement conditions using the following measuring instrument. Measuring instrument: Infinite 200 PRO M-Plex (manufactured by TECAN) Measurement conditions: Endpoint, wavelength 450 nm From the measured absorbance, the residual enzyme activity rate (%) was calculated by the following formula (1).

Number

[0030] <Example 1-2: Examples 1-2-1 to 1-2-4> A protein-stabilizing solution was prepared in the same manner as in Example 1-1, except that the compound of Synthesis Example 2 was used instead of the compound of Synthesis Example 1 and each concentration shown in Table 1 was used. Furthermore, the protein-stabilizing effect was evaluated in the same manner as in Example 1-1. The results are shown in Tables 1 to 4.

[0031] <Example 1-3: Examples 1-3-1 to 1-3-4> A protein-stabilizing solution was prepared in the same manner as in Example 1-1, except that the compound of Synthesis Example 3 was used instead of the compound of Synthesis Example 1 and each concentration shown in Table 1 was used. Furthermore, the protein-stabilizing effect was evaluated in the same manner as in Example 1-1. The results are shown in Tables 1 to 4.

[0032] <Example 1-4: Examples 1-4-1 to 1-4-5> A protein-stabilizing solution was prepared in the same manner as in Example 1-1, except that the compound of Synthesis Example 4 was used instead of the compound of Synthesis Example 1 and each concentration shown in Table 1 was used. Furthermore, the protein-stabilizing effect was evaluated in the same manner as in Example 1-1. The results are shown in Tables 1 to 4.

[0033] <Example 1-5: Examples 1-5-1 to 1-5-5> A protein-stabilizing solution was prepared in the same manner as in Example 1-1, except that the compound of Synthesis Example 5 was used instead of the compound of Synthesis Example 1 and each concentration shown in Table 1 was used. Furthermore, the protein-stabilizing effect was evaluated in the same manner as in Example 1-1. The results are shown in Tables 1 to 4.

[0034] <Example 1-6: Examples 1-6-1 to 1-6-5> A protein-stabilizing solution was prepared in the same manner as in Example 1-1, except that the compound of Synthesis Example 6 was used instead of the compound of Synthesis Example 1 and each concentration shown in Table 1 was used. Furthermore, the protein-stabilizing effect was evaluated in the same manner as in Example 1-1. The results are shown in Tables 1 to 4.

[0035] <Comparative Example 1-1: Comparative Examples 1-1-1 to 1-1-4> Using bovine serum albumin (BSA) as a protein stabilizer, the protein-stabilizing effect was evaluated in the same manner as in Example 1-1, except that each concentration shown in Table 1 was used. The results are shown in Tables 1 to 4.

[0036] <Comparative Example 1-2> The evaluation of the protein stabilization effect was carried out in the same manner as in Example 1-1, except that only phosphate buffer (pH = 7.4) was used without using a protein stabilizer. The results are shown in Tables 1 to 4.

[0037]

Table 1

[0038] From the measured absorbance (Table 1), the residual enzyme activity rate (%) was calculated by Equation (1). The results are shown in Table 2. When the residual activity rate after 600 days is 80% or more, it is judged as "◎" indicating an extremely high protein stabilization effect; when the residual activity rate is less than 60 - 80%, it is judged as "〇" indicating a high protein stabilization effect; when the residual activity rate is less than 60%, it is judged as "△" indicating a recognized protein stabilization effect.

[0039]

Table 2

[0040]

Table 3

[0041] From the measured absorbance (Table 3), the residual enzyme activity rate (%) was calculated by Equation (1). The results are shown in Table 4. When the residual activity rate after 550 days is 30% or more, it is judged as "◎" indicating an extremely high protein stabilization effect; when the residual activity rate is less than 10 - 30%, it is judged as "〇" indicating a high protein stabilization effect; when the residual activity rate is less than 10%, it is judged as "△" indicating a recognized protein stabilization effect.

[0042]

Table 4

[0043] As is clear from Tables 1 to 4, it can be seen that the protein stabilizers using the compounds according to the embodiments of the present invention in Examples 1-1 to 1-6 significantly stabilize the protein (peroxidase) compared to each comparative example. In particular, Examples 1-4 to 1-6 showed a higher protein stabilization effect at a lower concentration compared to other examples.

[0044] <Example 2-1> <Preparation of Protein Stabilizer Solution> The compound obtained in Synthesis Example 1 was dissolved in 1M Tris-HCl (pH 7.0) (manufactured by Nippon Gene Co., Ltd., hereinafter abbreviated as Tris buffer solution) to prepare a protein stabilizer solution having a final concentration twice the concentration shown in Tables 5 to 8.

[0045] <Evaluation of Protein Stabilization Effect> (1) An equal amount of the prepared protein stabilizer solution was mixed with a Tris buffer solution (protein solution) containing alkaline phosphatase (manufactured by Rockland Immunochemicals), sucrose (manufactured by Fujifilm Wako Pure Chemical Corporation), and MgCl2 (manufactured by Fujifilm Wako Pure Chemical Corporation) to prepare a protein stabilization solution. The alkaline phosphatase concentration in the protein stabilization solution was 0.2 μg / mL, the sucrose concentration was 10% by mass, and the MgCl2 concentration was 0.01 M. Therefore, the protein stabilization solution contained 0.1 μg / mL of alkaline phosphatase, and the concentration of the compound of Synthesis Example 1 in the protein stabilization solution was the concentration (% by mass) shown in Tables 5 to 8. (2) The protein stabilization solution was incubated at 25 °C or 37 °C for the number of days described in Tables 5 to 8. After incubation, it was added to a 96-well plate made of polystyrene at 8 μL / well, and 100 μL / well of a color-developing solution prepared by mixing equal amounts of Microwell phosphatase solution A (manufactured by LGC Clinical Diagnostics, Inc (SeraCare)) and Microwell phosphatase solution B (manufactured by LGC Clinical Diagnostics, Inc (SeraCare)) was added, and the color-developing reaction with the protein was carried out for 15 minutes. Subsequently, 50 μL / well of APstop Solution, 10× (manufactured by LGC Clinical Diagnostics, Inc (SeraCare)) was added to stop the color-developing reaction. The absorbance at a wavelength of 405 nm was measured, and the stabilization effect of alkaline phosphatase was evaluated. The absorbance was measured in the same manner as above except that the wavelength was 405 nm. The protein stabilization effect was evaluated by the residual enzyme activity rate (%) obtained from the above formula (1). The higher the value of the residual enzyme activity rate, the higher the protein stabilization effect. The evaluation results under the storage condition of 25 °C are shown in Tables 5 and 6, and the evaluation results under the storage condition of 37 °C are shown in Tables 7 and 8.

[0046] <Examples 2-2 to 2-6> Using the compounds of Synthesis Examples 2 to 6 instead of the compound of Synthesis Example 1, each protein stabilization solution was prepared in the same manner as in Example 2-1 so as to have the concentrations shown in Tables 5 to 8. Furthermore, the protein stabilization effect of each example was evaluated in the same manner as in Example 2-1. The results are shown in Tables 5 to 8.

[0047] <Comparative Example 2-1> The evaluation of the protein stabilization effect was carried out in the same manner as in Example 2-1 except that only Tris buffer (pH = 8.0) was used instead of the protein stabilizer solution. The results are shown in Tables 5 to 8.

[0048]

Table 5

[0049] From the measured absorbance (Table 5), the residual enzyme activity rate (%) was calculated by Equation (1). The results are shown in Table 6. When the residual activity rate after 300 days is 40% or more, it is judged as "◎" indicating an extremely high protein stabilization effect; when the residual activity rate is less than 10 - 40%, it is judged as "〇" indicating a high protein stabilization effect; when the residual activity rate is less than 10%, it is judged as "△" indicating a protein stabilization effect.

[0050]

Table 6

[0051]

Table 7

[0052] From the measured absorbance (Table 7), the residual enzyme activity rate (%) was calculated by Equation (1). The results are shown in Table 8. When the residual activity rate after 50 days is 20% or more, it is judged as "◎" indicating an extremely high protein stabilization effect; when the residual activity rate is less than 10 - 20%, it is judged as "〇" indicating a high protein stabilization effect; when the residual activity rate is less than 10%, it is judged as "△" indicating a protein stabilization effect.

[0053]

Table 8

[0054] As is clear from Tables 5 - 8, it can be seen that the protein stabilizer using the compounds according to the embodiments of the present invention in Examples 2 - 1 to 2 - 6 stabilizes the protein (alkaline phosphatase) as compared with Comparative Example 2 - 1.

Claims

1. A protein stabilizer containing a compound represented by formula (1) as an active ingredient. 【Chemical 1】 [In formula (1), n is an integer from 5 to 15. ]

2. The protein stabilizer according to claim 1, which contains water.

3. The protein stabilizer according to claim 1 or 2, wherein the protein to be stabilized is at least one of peroxidase and alkaline phosphatase.

4. A method for stabilizing a protein, which comprises co-existing the protein and the compound represented by formula (1) in a solution containing water. 【Chemical 2】 [In formula (1), n is an integer from 5 to 15. ]

Images