Protein stabilizing agents, kits, and methods of protecting proteins

Abstract

The application discloses a protein stabilizer, a kit and a method for protecting a protein. In the application, the protein stabilizer comprises 1-10 parts by weight of bovine serum albumin (BSA), 0.1-5 parts by weight of a non-ionic surfactant, 5-40 parts by weight of a polyhydric alcohol, 1-10 parts by weight of other additives and 35-92.9 parts by weight of water, wherein the other additives are at least one selected from a saccharide additive, polyvinylpyrrolidone and carbonyldiamide. The protein stabilizer provided by the application has simple formula, safe components, low manufacturing cost and wide application range; the protein added with the protein stabilizer can be stored at room temperature, repeated freezing and thawing and sub-packaging are avoided, and the protein is convenient to use.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and particularly to protein stabilizers, reagent kits, and methods for protecting proteins. Background Technology

[0002] Protein products, such as antigens and antibodies, are prone to deterioration and inactivation when stored at room temperature. To extend the shelf life of proteins, current technologies often employ methods such as low-temperature storage, freeze-drying, increasing protein concentration, and adding protease inhibitors to maintain protein activity for a longer period. Low-temperature storage involves placing proteins in a low-temperature freezer, which can increase their stability, but requires repeated freeze-thaw cycles before use. Repeated freeze-thaw cycles can denature antibodies, causing them to aggregate and reducing their binding capacity, making them more susceptible to degradation. Freeze-drying involves using a freeze dryer to freeze-dry proteins into powder for storage. This method requires large-scale freeze-drying equipment consisting of a drying chamber, refrigeration system, vacuum system, heating system, and control system, which is expensive and time-consuming (approximately 2-3 days) to convert the protein into powder. Furthermore, low-temperature dehydration makes it difficult to purify some microorganisms, potentially leaving bacteria and cold-resistant pathogens in the product. Even slight contact with moisture during storage and packaging can cause protein inactivation. Increasing protein concentration requires purifying and concentrating the protein, which can slow down protein deterioration to some extent, but the operation is cumbersome, and purification and concentration conditions are difficult to control. Protease inhibitors are toxic and harmful to health.

[0003] To overcome the shortcomings of methods such as low-temperature storage, freeze-drying, increasing protein concentration, and adding protease inhibitors, various protein stabilizers are often added to protein products. Chinese Patent CN106199007B discloses a protein protectant comprising 0.318% phosphate (sodium dihydrogen phosphate), 0.5% bovine serum albumin, 0.2% Tween, and combinations of one or more selected from adenosine monophosphate (AMP), sodium heparin, MgCl2, polyvinylpyrrolidone 40 (PVP40), tea polyphenols, cyclodextrin, and K2SO4. This protectant has a complex composition and only shows good stabilization effect on Hsp90α protein, lacking universality. Furthermore, most commercially available protein stabilizers have complex formulations and poor stability, failing to maintain protein stability under extreme conditions such as high temperature, repeated freeze-thaw cycles, and dilution. Therefore, there is a need in the field to find a protein stabilizer with a simple formulation, low cost, and good stabilization effect for various proteins. Summary of the Invention

[0004] The purpose of this invention is to provide a protein stabilizer that has a simple formulation, low cost, and is easy to use. It can universally stabilize a variety of proteins and has a good stabilizing effect.

[0005] To address the aforementioned technical problems, a first aspect of the present invention provides a protein stabilizer, the protein stabilizer comprising:

[0006]

[0007] The other additives are selected from at least one of sugar additives, polyvinylpyrrolidone, and carbodiamine.

[0008] In some preferred embodiments, the nonionic surfactant is selected from at least one of Triton, polysorbate (Tween 80), polyoxyethylene fatty acid ester, lecithin, and poloxamer, more preferably Triton.

[0009] In some preferred embodiments, the polyol is selected from at least one of glycerol and propylene glycol, more preferably glycerol.

[0010] In some preferred embodiments, the sugar additive is selected from at least one of sucrose, chitosan, trehalose and methyl glucose polyether, more preferably sucrose or trehalose.

[0011] In some preferred embodiments, the protein stabilizer comprises:

[0012]

[0013] In some preferred embodiments, the protein stabilizer is composed of:

[0014]

[0015] In some preferred embodiments, the protein stabilizer is composed of:

[0016]

[0017] In some preferred embodiments, the protein stabilizer is composed of

[0018]

[0019] In some preferred embodiments, the protein is selected from at least one of NGAL antigen, β2-MG antigen, TRF antigen, RBP antigen, SAA monoclonal antibody, PG2 monoclonal antibody, PG1 monoclonal antibody and FSH monoclonal antibody.

[0020] A second aspect of the present invention provides a method for stabilizing proteins in a liquid sample, the method comprising the steps of:

[0021] The protein stabilizer described in the first aspect of the present invention is mixed with a liquid sample containing the protein to be protected.

[0022] In some preferred embodiments, the molar concentration of the protein to be protected protein in the mixture of the protein stabilizer and the liquid sample containing the protein to be protected is 0.05 to 0.3 μg / mL, more preferably 0.1 to 0.2 μg / mL, for example 0.15 μg / mL.

[0023] A third aspect of the present invention provides a kit comprising the protein stabilizer described in the first aspect of the present invention.

[0024] Compared with the prior art, the present invention has at least the following advantages:

[0025] (1) The protein stabilizer provided by the present invention has a simple formulation, safe components, low manufacturing cost, and wide applicability;

[0026] (2) The protein stabilizer provided by the present invention has a good effect on stabilizing proteins. Proteins with the protein stabilizer provided by the present invention can be stored at room temperature, avoiding repeated freeze-thaw cycles and repackaging.

[0027] (3) The protein stabilizer provided by the present invention is easy to use; it can be directly mixed with the protein sample to be protected.

[0028] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0029] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0030] Figure 1 This is a graph showing the experimental results of stabilizing the NGAL antigen with the protein stabilizer in Test Example 1 according to the present invention;

[0031] Figure 2 This is a graph showing the experimental results of stabilizing the RBP antigen with a protein stabilizer in Test Example 2 of this invention. Detailed Implementation

[0032] The inventors discovered in their research that existing protein stabilizers have limited applicability, complex formulations, and poor protein stabilization effects. Therefore, through extensive experimentation, the inventors screened a protein stabilizer comprising: 1%–10% bovine serum albumin (BSA); 0.1%–5% nonionic surfactant; 5%–40% polyol; 1%–10% other additives; and water, wherein the other additives are selected from at least one of carbohydrate additives, polyvinylpyrrolidone, and carbodiamine. This protein stabilizer not only has a simple formulation and low preparation cost, but also can stabilize various proteins including antigens and antibodies, and exhibits excellent stabilization effects on various proteins, thus completing this invention.

[0033] Furthermore, the inventors optimized the formula, using Triton as the nonionic surfactant and selecting sugar additives, especially sucrose and trehalose, which resulted in the best protein stabilization effect.

[0034] The "substrate stabilizer CCD" and "antibody stabilizer DS" used in this article are commercially available broad-spectrum protein stabilizers.

[0035] In some preferred embodiments of the present invention, the protein stabilizer comprises:

[0036]

[0037] The other additives are selected from at least one of sugar additives, polyvinylpyrrolidone, and carbodiamine.

[0038] In some preferred embodiments, the nonionic surfactant is selected from at least one of Triton, polysorbate (Tween 80), polyoxyethylene fatty acid ester, lecithin, and poloxamer, more preferably Triton.

[0039] In some preferred embodiments, the polyol is selected from at least one of glycerol and propylene glycol, more preferably glycerol.

[0040] In some preferred embodiments, the sugar additive is selected from at least one of sucrose, chitosan, trehalose and methyl glucose polyether, more preferably sucrose or trehalose.

[0041] In some preferred embodiments, the protein stabilizer comprises:

[0042]

[0043] In some preferred embodiments, the protein stabilizer is composed of:

[0044]

[0045]

[0046] In some preferred embodiments, the protein stabilizer is composed of:

[0047]

[0048] In some preferred embodiments, the protein stabilizer is composed of

[0049]

[0050] In some preferred embodiments, the protein is selected from at least one of NGAL antigen, β2-MG antigen, TRF antigen, RBP antigen, SAA monoclonal antibody, PG2 monoclonal antibody, PG1 monoclonal antibody and FSH monoclonal antibody.

[0051] In some preferred embodiments of the present invention, a method for stabilizing proteins in a liquid sample is provided, the method comprising the steps of:

[0052] The protein stabilizer described in the first aspect of the present invention is mixed with a liquid sample containing the protein to be protected.

[0053] In some preferred embodiments, the molar concentration of the protein to be protected protein in the mixture of the protein stabilizer and the liquid sample containing the protein to be protected is 0.05 to 0.3 μg / mL, more preferably 0.1 to 0.2 μg / mL, for example 0.15 μg / mL.

[0054] In some preferred embodiments of the present invention, a kit is provided comprising the protein stabilizer described in the first aspect of the present invention.

[0055] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0056] Unless otherwise specified, the 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 pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0057] Example 1: Preparation of protein stabilizer

[0058] Bovine serum albumin (BSA), triaton, glycerol, and sucrose are mixed thoroughly to prepare a protein stabilizer. The mass fractions of each component are as follows:

[0059]

[0060] Example 2: Preparation of protein stabilizer

[0061] Bovine serum albumin (BSA), triaton, glycerol, and sucrose are mixed thoroughly to prepare a protein stabilizer. The mass fractions of each component are as follows:

[0062]

[0063] Example 3: Preparation of protein stabilizer

[0064] Bovine serum albumin (BSA), triaton, glycerol, and sucrose are mixed thoroughly to prepare a protein stabilizer. The mass fractions of each component are as follows:

[0065]

[0066] Example 4: Preparation of protein stabilizers

[0067] Bovine serum albumin (BSA), triaton, glycerol, and sucrose are mixed thoroughly to prepare a protein stabilizer. The mass fractions of each component are as follows:

[0068]

[0069] Example 5: Preparation of protein stabilizer

[0070] Bovine serum albumin (BSA), triaton, glycerol, and sucrose are mixed thoroughly to prepare a protein stabilizer. The mass fractions of each component are as follows:

[0071]

[0072] Comparative Example 1: Preparation of Protein Stabilizer

[0073] Bovine serum albumin (BSA), Tween 20, glycerol, and sucrose are mixed thoroughly to prepare a protein stabilizer. The mass fractions of each component are as follows:

[0074]

[0075] Comparative Example 2: Preparation of Protein Stabilizer

[0076] Bovine serum albumin (BSA), triaton, glycerol, and sucrose were mixed thoroughly. The mass fractions of each component are as follows:

[0077]

[0078]

[0079] Test Example 1: Experiment on stabilizing NGAL antigen with protein stabilizers

[0080] Preparation of NGAL antigen: The preparation of NGAL antigen was carried out in accordance with the method described in the reference "Human NGAL Cloning Expression and Antibody Preparation and Identification [J]. Journal of Chongqing Medical University, 2019, 44(05): 555-560." Specifically, the NGAL antigen was artificially synthesized by codon optimization of the NGAL gene, the prokaryotic expression recombinant plasmid pET28a-NGAL was constructed, and after IPTG induction expression, the plasmid was isolated, purified and analyzed in solution to obtain the NGAL antigen.

[0081] Experimental group: NGAL antigen was taken and diluted with substrate stabilizer CCD, antibody stabilizer DS, and protein stabilizer prepared in Example 1, respectively, to an antigen concentration of 0.15 μg / mL.

[0082] Control group: Substrate stabilizer CCD, antibody stabilizer DS, and protein stabilizer prepared in Example 1 were used as blank control group.

[0083] The Tm and Tagg proteins of the experimental and control groups were measured using the Uncle multifunctional protein stability analysis system, and plotted... Figure 1 Among them, Tagg 实验组 -Tagg 对照组 =Tagg, calculate the Tagg value and record it in Table 1.

[0084] Table 1

[0085] 1 Example 1 88.0 2 stabilizer CCD 85.7 3 stabilizer AS 65.0

[0086] According to Table 1 and Figure 1 The results showed that the NGAL antigen with the protein stabilizer prepared in Example 1 had better stability and was less prone to aggregation.

[0087] Test Example 2: Experiment on stabilizing RBP antigen with protein stabilizers

[0088] RBP antigen preparation: The preparation of RBP antigen was based on the method described in the reference "Expression of human retinol-binding protein prokaryotic protein and preparation of rabbit polyclonal antibody [J]. Journal of Cellular and Molecular Immunology, 2019, 35(07):653-658." Specifically, the human RBP gene was amplified by reverse transcription PCR, and the amplified product was ligated into the prokaryotic expression vector pET-28a(+) to construct the recombinant plasmid pET-28a(+)-RBP, which was then transformed into Escherichia coli. Expression was induced by isopropyl-β-D-thiogalactoside, and the expression product was identified as a positive clone by SDS-PAGE analysis. The culture was expanded, and the recombinant RBP was purified using a histone tag purification column.

[0089] Experimental group: RBP antigen was taken and diluted with stabilizer CCD, stabilizer DS and protein stabilizer prepared in Example 1 respectively, until the antigen concentration was 0.15 μg / mL.

[0090] Control group: Stabilizer CCD, stabilizer DS, and protein stabilizer prepared in Example 1 were used as blank control group.

[0091] The Tm and Tagg proteins of the experimental and control groups were measured using the Uncle multifunctional protein stability analysis system, and plotted... Figure 2 Among them, Tagg 实验组 -Tagg 对照组 =Tagg, calculate the Tagg value and record it in Table 2.

[0092] Table 2

[0093] 1 Example 1 88.8 2 stabilizer CCD 81.8 3 stabilizer DS 71.2

[0094] According to Table 2 and Figure 2 The results showed that the RBP antigen with the protein stabilizer prepared in Example 1 had better stability and was less prone to aggregation.

[0095] Test Example 3: Accelerated Protein Stability Experiment

[0096] Preparation of β2-MG antigen: The preparation of β2-MG antigen refers to the method described in the literature "Optimization and purification of prokaryotic expression conditions of human β2 microglobulin [J]. Journal of Biomedical Engineering, 2015, 32(05): 1050-1055." Specifically, the recombinant plasmid pET32a was constructed, and the antigen was obtained by induction expression in Escherichia coli and then isolated and purified.

[0097] TRF antigen preparation: The preparation of TRF antigen refers to the method described in the literature "Cloning, expression and antibody preparation of human telomere-binding protein TRF1 [J]. Chinese Journal of Biotechnology, 2004(01):30-33.4." Specifically, the His6-TRF fusion protein was induced by IPTG to construct the His6-tag prokaryotic expression vector pET-28a-TRF to obtain the TRF antigen.

[0098] Preparation of SAA monoclonal antibody, PG2 monoclonal antibody, PG1 monoclonal antibody and FSH monoclonal antibody: expression vectors were constructed, and CHO suspension cells were used to express and culture them. The cell supernatant was collected, separated and purified to obtain the corresponding antibodies.

[0099] NGAL, β2-MG, TRF, RBP antigens and SAA, PG2, PG1, and FSH monoclonal antibodies were diluted with the protein stabilizer prepared in Example 1 to a concentration of 0.15 μg / mL for each antigen and monoclonal antibody. The solutions were incubated at 37°C for 7 days, and samples were taken on days 4 and 7 to measure their stability.

[0100] The Tm and Tagg of each antigen and monoclonal antibody were measured using the UNcle multifunctional protein stability analysis system. Tagg was calculated after subtracting the blank. The results are shown in Table 3.

[0101] Table 3

[0102]

[0103]

[0104] As shown in Table 3, compared to the original antigen and antibody solutions, the Tagg values ​​of the antigen and antibody after adding the protein stabilizer of Example 1 of this invention significantly increased at 37°C for 4 and 7 days, indicating that the stability of both the antigen and antibody was greatly enhanced after adding the protein stabilizer of Example 1 of this invention. The protein stabilizer of Example 1 of this invention is universally applicable to many proteins, such as antigens and antibodies. Preferably, the protein stabilizer of Example 1 of this invention is suitable for NGAL antigen, β2-MG antigen, TRF antigen, RBP antigen, SAA monoclonal antibody, PG2 monoclonal antibody, PG1 monoclonal antibody, and FSH monoclonal antibody.

[0105] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. A protein stabilizer, characterized in that, The protein is selected from at least one of NGAL antigen, β2-MG antigen, TRF antigen, RBP antigen, SAA monoclonal antibody, PG2 monoclonal antibody, PG1 monoclonal antibody and FSH monoclonal antibody; The protein stabilizer consists of the following components: Bovine serum albumin (BSA) 1-5 parts by weight; Triton 0.5~1.5 parts by weight; 10-25 parts by weight of glycerin; 3-7 parts by weight of sucrose; and Water 61.5~85.5 parts by weight.

2. A method for stabilizing proteins in a liquid sample, characterized in that, The method includes the following steps: Mix the protein stabilizer as described in claim 1 with a liquid sample containing the protein to be protected; The protein to be protected is selected from at least one of NGAL antigen, β2-MG antigen, TRF antigen, RBP antigen, SAA monoclonal antibody, PG2 monoclonal antibody, PG1 monoclonal antibody and FSH monoclonal antibody.

3. The method according to claim 2, characterized in that, In the mixture of the protein stabilizer and the liquid sample containing the protein to be protected, the molar concentration of the protein to be protected is 0.05~0.3 μg / mL.

4. A reagent kit, characterized in that, The kit includes the protein stabilizer as described in claim 1.

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