A process for extracting high-purity amelogenin

Abstract

The invention discloses a high-purity amelogenin extraction process, comprising the following steps: S1. providing a crude extract of enamel matrix protein; S2. adding a trichloroacetic acid solution to the crude extract of enamel matrix protein, shaking and incubating, so that the precipitate is fully precipitated; wherein the concentration of trichloroacetic acid in the crude extract of enamel matrix protein is 1.35%≤c≤1.50%; S3. centrifuging the incubated mixed solution and collecting the supernatant; S4. continuing to add the trichloroacetic acid solution to the collected supernatant, shaking and incubating; after the precipitate is fully precipitated, centrifuging and collecting the precipitate to obtain amelogenin P148. The high-purity amelogenin extraction process provided by the invention has simple steps, good repeatability, high purification efficiency and low cost.

Description

Technical Field

[0001] The invention relates to the technical field of protein extraction technology, and in particular to a process for extracting high-purity amelogenin. Background Art

[0002] Protein is the functional executor of many life activities, and its related research is one of the hot spots in life science research. Among them, enamel matrix protein is a family of proteins extracted from the developing tooth enamel. Its composition is complex and diverse, containing dozens of protein components mainly from the amelogenin family. These amelogenins have similar molecular segments and hydrophilicity. Studies have found that enamel matrix protein shows multi-functional therapeutic effects in periodontal regeneration and is the most prestigious periodontal therapeutic growth factor. It has achieved good clinical results in the treatment of periodontal and gingival regeneration, apical closure, pulp capping, peri-implantitis and soft tissue wound healing. The product has a very low cost (extracted from unerupted molars of pig mandibles), but the sales price is expensive (70 mg is priced at nearly US$500). The enamel matrix protein product developed based on empirical science has been sold well in many countries around the world for more than 20 years. However, despite the significant clinical therapeutic effect of enamel matrix protein, its related mechanism of periodontal regeneration is still unclear and so far. Emdogain is an enamel matrix protein drug launched by dental giant Straumann, and is the only low-immunogenicity biological agent on the market for periodontal treatment. Emdogain is not currently available in China, and domestic pharmaceutical companies have not yet developed enamel matrix protein-based drugs similar to Emdogain.

[0003] Purification of proteins is an important prerequisite for conducting research on protein structure and function. During the experiment, solvent extraction is generally used to release proteins from original tissues and cells in a dissolved state. The protein solution extracted from the raw material generally has a low protein content and contains a variety of impurities. Therefore, the extract is initially extracted, also known as crude extraction or crude fractionation, with the main purpose of removing sugars, lipids, nucleic acids and most impurities. The crude separation operation should generally be as simple and fast as possible, and is suitable for processing a large number of samples, so the precipitation method is mainly used. Precipitation is the process in which the solute in the solution changes from liquid phase to solid phase. Combined with other solid-liquid separation techniques such as centrifugal filtration, it can achieve the purpose of rapid concentration and separation of proteins. Common methods for protein precipitation include salting out, isoelectric precipitation, organic solvent precipitation, alkaloid reagent and certain acid (such as trichloroacetic acid) precipitation, etc. Precipitation techniques include simple precipitation, graded precipitation, etc. Graded precipitation is a method of adding precipitants in batches to precipitate different proteins at different precipitant concentrations, thereby separating them.

[0004] The purification of enamel matrix protein is a difficult problem that troubles many materials science and oral research workers. First, enamel matrix protein is a natural biological drug extracted from the enamel of the developing period, with complex ingredients, obvious batch differences, and difficult to evaluate the quality of the finished product. Studies have found that Emdogain contains many amelogenin components such as P173, P161, P148, TRAP (tyrosine-rich amelogenin peptide), 4.5K, 4.3K, 3K, etc., among which the most important proteins are TRAP, P148, P173 and P161. Second, it is difficult to purify amelogenin monomers, which are the main components of enamel matrix protein. This has led to a lack of amelogenin monomers as a research material in China, making it difficult to conduct precise pharmacological and pharmacodynamic studies on enamel matrix proteins. This has led to the fact that the periodontal regeneration mechanism of enamel matrix protein is still unclear, further exacerbating the in-depth research and development of enamel matrix protein products.

[0005] At present, the scheme for extracting crude amelogenin from the tooth enamel tissue of animals during their growth and development period is already very mature. The research work on separating and purifying natural amelogenin components from crude amelogenin has been carried out both at home and abroad, and the main purification method used is a combination of gel permeation chromatography and reversed-phase liquid chromatography. However, the existing methods generally have problems such as low purity of the obtained amelogenin product, long purification cycle, large sample loss, low repeatability of the purification process, and high cost.

[0006] Therefore, developing a method for purifying enamel matrix protein with simple steps and the ability to obtain high-purity amelogenin monomers without the need for chromatography is of great significance for the development of domestic new enamel matrix protein drugs. Summary of the invention

[0007] The present invention aims to solve the problem that amelogenin monomer is difficult to purify and obtain, and provides a process for extracting high-purity amelogenin, which has simple steps, good repeatability, high purification efficiency and low cost.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] The present invention provides a process for extracting high-purity amelogenin, comprising the following steps:

[0010] S1. Provide crude extract of enamel matrix protein;

[0011] S2. adding trichloroacetic acid solution to the crude extract of enamel matrix protein, shaking and incubating, so that the precipitate is fully precipitated; wherein the concentration of trichloroacetic acid in the crude extract of enamel matrix protein is 1.35%≤c≤1.50%;

[0012] S3. Centrifuging the incubated mixture and collecting the supernatant;

[0013] S4. Continue to add trichloroacetic acid solution to the collected supernatant, shake and incubate; after the precipitate is fully precipitated, centrifuge and collect the precipitate to obtain the target amelogenin P148.

[0014] Trichloroacetic acid is a common protein precipitation method and is often used for crude protein extraction. The inventor found in previous studies that trichloroacetic acid also has the effect of graded precipitation. Therefore, the inventor used this property of trichloroacetic acid to graded enamel matrix protein and finally separated and obtained high-purity amelogenin P148. Specifically, several important proteins in enamel matrix protein - TRAP, P148, P173 and P161 - will precipitate in the presence of low concentrations of trichloroacetic acid, but when the concentration of trichloroacetic acid exceeds a certain concentration, the precipitation amount of TRAP, P173 and P161 is close to 0, while P148 will still have a considerable amount of precipitation, so this part of the precipitation is collected to obtain high-purity P148. Compared with the amelogenin separation and purification method in the prior art, the method provided by the present invention is very simple, does not require auxiliary means such as chromatography, simplifies the purification steps, and reduces the cost of purification. At the same time, the method can be used for mass production of high-purity P148 protein, and has a good industrialization prospect.

[0015] In step S1 of the present invention, the crude extract of enamel matrix protein can be obtained by extracting from tooth enamel using an extracting solution, or by dissolving the crude amelogenin product in an organic solvent. The extracting solution or organic solvent is preferably an acetic acid solution. Preferably, the crude extract of enamel matrix protein is a saturated solution of enamel matrix protein.

[0016] In step S2 of the present invention, the trichloroacetic acid solution is preferably added to the crude extract of enamel matrix protein by dropwise addition, so that the protein can be fully precipitated. The concentration of the trichloroacetic acid solution is not limited, preferably 10% to 20%, for example, it can be 10%, 12%, 15%, 16%, 18%, 20%, etc. It should be pointed out that the concentration of trichloroacetic acid here is the mass volume concentration. Taking the preparation of trichloroacetic acid with a concentration of 10% as an example, 10g of trichloroacetic acid is weighed, dissolved in water, and then fixed to 100mL. The resulting solution is a 10% trichloroacetic acid aqueous solution.

[0017] In step S2 of the present invention, after adding trichloroacetic acid solution to the crude extract of enamel matrix protein, it is necessary to ensure that the concentration of trichloroacetic acid in the solution c≥1.35%. When c≥1.35%, the precipitated protein is mostly P148, and the precipitation amount of other proteins is almost 0. However, the concentration of trichloroacetic acid should not be too high. If the concentration c>1.40%, most of the P148 in the crude extract of enamel matrix protein is precipitated, resulting in a low yield of the separated and purified P148. Preferably, the concentration c of trichloroacetic acid satisfies: 1.35%≤c≤1.40%. For example, c can be 1.35%, 1.36%, 1.37%, 1.38%, 1.39%, 1.40%, and so on.

[0018] Furthermore, in step S2, the incubation time is 1 to 5 minutes. After the trichloroacetic acid solution is added, the three types of proteins TRAP, P173 and P161 can be fully precipitated through incubation to avoid precipitation when trichloroacetic acid is added later, thereby avoiding affecting the purity of amelogenin P148.

[0019] Furthermore, in step S3, the centrifugal force of the centrifugation is 3000-5000 g, and the centrifugation time is 3-10 min.

[0020] In step S4 of the present invention, after the trichloroacetic acid solution is continuously added to the supernatant, the concentration of trichloroacetic acid in the system is further increased, so the P148 protein continues to precipitate, while the other three proteins are hardly precipitated, so that the components in the precipitate are almost all P148. However, when the concentration c of trichloroacetic acid exceeds 1.70%, the amount of P148 precipitated is very small, so preferably, after the trichloroacetic acid solution is added, the concentration c of trichloroacetic acid in the solution is controlled to be ≤1.7%.

[0021] Furthermore, in step S4, the collected precipitate is resuspended in a solvent to remove residual trichloroacetic acid, and then the solvent is removed by centrifugation, and the mixture is allowed to stand and dry to obtain pure white P148 protein powder.

[0022] Furthermore, in step S4, the solvent is ethanol.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention utilizes the characteristic of trichloroacetic acid having the effect of graded precipitation, and controls trichloroacetic acid of different concentrations to graded precipitate enamel matrix protein, and finally separates high-purity amelogenin P148 from the crude extract of enamel matrix protein. The extraction process is simple and feasible, and no chromatographic auxiliary means such as gel permeation chromatography and reversed-phase liquid chromatography are required, which greatly simplifies the purification process and reduces the cost of purification. In addition, the method has good reproducibility and can be used for mass production of high-purity P148 protein, and has good industrialization prospects.

[0025] 2. The protein component P148 separated by the present invention has a very high purity and can eliminate the influence of other protein components. It is suitable for the research on the mechanism of enamel mineralization and material bionics research and the development and production of periodontal regeneration drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A process for precipitating an acetate protein solution using trichloroacetic acid;

[0027] Figure 2 The peak shape of the chromatographic elution curve of precipitates 1 to 4;

[0028] Figure 3 The peak shape of the chromatographic elution curve is 5 to 8 of precipitation;

[0029] Figure 4 is the electrophoretogram of the product extracted in Example 2;

[0030] Figure 5 It is the mass spectrum of the product extracted in Example 2. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0033] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0034] 1. Instruments and Reagents

[0035] 1. Instrument

[0036] Agilent 1100 high performance liquid chromatography, high-speed centrifuge (model: TG16.5), 10 mL horizontal rotor, conical centrifuge tubes.

[0037] 2. Reagents

[0038] Unerupted pig molar (provided by the laboratory); trichloroacetic acid (99%, Adamas Reagent Co., Ltd.); glacial acetic acid (analytical grade, Guangzhou Chemical Reagent Co., Ltd.); acetone (analytical grade, Guangzhou Chemical Reagent Co., Ltd.); acetonitrile (Adamas-beta HPLC grade); and pure water (Millipore).

[0039] 2. Experimentation and Characterization

[0040] Example 1

[0041] 1. Preparation of Enamel Matrix Protein Samples

[0042] 1.1 Preparation of TCA precipitation supernatant

[0043] Unerupted wisdom teeth were dug out from the mandible of the pig, blood stains were washed off, loose enamel rich in organic matter was scraped with a blade, and extracted with 0.5M acetic acid at low temperature for 24 hours. Solid impurities in the acetate protein solution were removed by centrifugation to obtain enamel matrix protein acetate solution.

[0044] 1.2 Protein precipitation by trichloroacetic acid method

[0045] The acetate protein solution was precipitated using the trichloroacetic acid method. The steps are as follows: Figure 1 , the specific method is: take 8mL of enamel matrix protein solution, place it in a 10mL conical bottom centrifuge tube, and use the centrifuge tube as a container for protein precipitation. During the first trichloroacetic acid precipitation operation, use a pipette to add 300 microliters of 15% trichloroacetic acid solution. As the trichloroacetic acid liquid flow is added, white protein precipitates can be seen wherever the liquid flows. Shake the centrifuge tube slightly and incubate for 1 minute. Cover the centrifuge tube cap, centrifuge at 4000g for 5 minutes in a horizontal rotor, collect the supernatant and transfer it to another centrifuge tube, then add 100 microliters of 15% trichloroacetic acid to the supernatant again, centrifuge to obtain precipitate 2; transfer the supernatant to another centrifuge tube, add 100 microliters of 15% trichloroacetic acid again, centrifuge to obtain precipitate 3, and so on, to obtain 8 precipitates. They correspond to the enamel matrix protein precipitates corresponding to trichloroacetic acid under different concentration gradients.

[0046] 2. Characterization of Enamel Matrix Protein Precipitates

[0047] 2.1 HPLC chromatographic analysis of protein precipitation

[0048] Resuspend the 8 precipitates with ethanol, wash the remaining trichloroacetic acid, centrifuge to remove the ethanol, let stand to evaporate and dry, add 800μL of pure water to each tube, so that all 8 precipitate samples are completely dissolved. Add to the sample bottle for HPLC analysis. The injection volume is 100μL, and 220nm is set as the monitoring wavelength. The chromatographic system used for all samples is consistent, and the peaks of several important components are separated as much as possible to facilitate the comparison of the trichloroacetic acid precipitation effect.

[0049] 2.2 Results and Discussion

[0050] It was observed that the 8 protein precipitates obtained could all be completely re-dissolved in pure water, and the so-called phenomenon of trichloroacetic acid completely destroying the protein structure was not observed.

[0051] The most important proteins in the enamel matrix proteins are TRAP, P148, P173 and P161. Their contents and relative proportions can be used to clearly evaluate the effect of trichloroacetic acid precipitation. The chromatograms of the eight precipitations obtained are listed in Figure 2 and Figure 3 In the chromatogram, the known important components have been identified. From the chromatogram, it can be seen that because a consistent chromatographic analysis system is adopted, the retention times of TRAP, P148, P173 and P161 in different HPLC chromatograms are highly stable. Figure 3 and Figure 4 From the peak shape of the chromatographic elution curve, we can see that the relative proportions and masses of several typical protein components obtained in different precipitation stages have been changing. This alone directly shows that the trichloroacetic acid precipitation method is also a typical graded precipitation method. By controlling the concentration of trichloroacetic acid, different components of protein precipitates can be precipitated out from the mixed solution step by step to achieve the effect of separating the target protein, which can provide the possibility for segmented cutting, directional purification and component matching of the total protein.

[0052] The peak area is proportional to the amount of precipitated protein in this section. The peak area of ​​each protein was integrated using the Chemstation software provided by Agilent 1200. The peak area is shown in Table 1.

[0053] Table 1 Peak area of ​​each precipitated protein signal peak

[0054] TRAP P148 P173 P161 Precipitation 1 (0-300μL) 29121.6 14118 3915.8 3189.4 Precipitation II (300-400 μL) 39608.2 33913 6009.2 5545.8 Precipitation III (400-500 μL) 22790.7 44754.9 3700.9 4375.4 Precipitation IV (500-600 μL) 15172.8 54125 2263 2272.7 Precipitation 5 (600-700 μL) 1873.9 17944.3 333.7 175 Precipitation VI (700-800 μL) 507 15277.8 132 --- Precipitation VII (800-900 μL) --- 10973 --- --- Precipitation 8 (900-1000μL) --- 5919.8 --- ---

[0055] From the results and trend analysis of trichloroacetic acid precipitation, we can get:

[0056] ① In the 0-300μL process, the peak areas of TRAP, P148, P173 and P161 were 29121.6, 14118, 3915.8 and 3189.4 respectively. In the 300-400μL section, the peak areas of TRAP, P148, P173 and P161 increased to 39608.2, 33913, 6009.2 and 5545.8 respectively. This shows that the precipitation effect can only be exerted after the trichloroacetic acid precipitation reaches a certain concentration.

[0057] ②In the 400-500μL stage, the peak areas of TRAP, P173 and P161 were 22790, 3700.9 and 4375.4, showing a downward trend compared with the 300-400μL stage, and this downward trend has been maintained. This shows that these proteins are sensitive to lower concentrations of trichloroacetic acid, while the peak area of ​​P148 continues to grow and reaches a peak in the 500-600μL trichloroacetic acid range. Regardless of the type of protein, the amount of precipitation increases with the increase of trichloroacetic acid concentration in the solution, showing an upward trend in the early stage and a downward trend in the later stage. This situation clearly shows that different types of proteins have different corresponding trichloroacetic acid concentrations when they reach the maximum amount of precipitation.

[0058] ③At the 800-900μL stage, the peak areas of TRAP, P173 and P161 are close to zero, while the peak area of ​​P148 is 10973, indicating that the protein precipitate obtained at this time is high-purity P148. This result can also be verified in the visual perception of the precipitate, because the early protein precipitate is light yellow, and at the 800-900μL stage, the precipitate is pure white, which is also the color characteristic of pure P148 protein powder. This result suggests that it is possible to obtain a certain high-purity protein from a complex protein system using only the simple precipitation method of trichloroacetic acid precipitation.

[0059] Example 2

[0060] Take 8 mL of the enamel matrix protein solution prepared in Example 1 and place it in a 10 mL conical bottom centrifuge tube. Use a pipette to add 800 microliters of 15% trichloroacetic acid solution (after addition, the concentration of trichloroacetic acid in the enamel matrix protein solution is 1.36%). As the trichloroacetic acid liquid flow is added, white protein precipitation can be seen wherever the liquid flow reaches. Shake the centrifuge tube slightly and incubate for 3 minutes. Cover the centrifuge tube cover, centrifuge at 4000g for 5 minutes in a horizontal rotor, collect the supernatant and transfer it to another centrifuge tube. Then add 100 microliters of 15% trichloroacetic acid to the supernatant again (after addition, the concentration of trichloroacetic acid in the supernatant is 1.52%), and centrifuge to obtain a precipitate. Use ethanol to resuspend the precipitate, wash the remaining trichloroacetic acid, centrifuge to remove the ethanol, and then stand to evaporate and dry to obtain a pure white P148 protein powder.

[0061] Figure 4 and Figure 5 They are respectively the electrophoretogram and mass spectrogram of the P148 protein precipitate extracted in Example 2. As can be seen from the electrophoretogram, the content of impurity protein in the precipitate is very small. It can also be seen from the mass spectrogram that the impurity content in the precipitate is very low (few impurity peaks), and there are only two signal peaks caused by one molecule, wherein 16875.666 represents that P148 has one charge, and 8451.792 represents that P148 has two charges (double electric peaks). Both peaks are signals generated by the P148 molecule, which proves that the precipitate finally obtained is the P148 protein.

[0062] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A process for extracting high-purity amelogenin, It is characterized in that The following steps are involved: S1. Providing a crude extract of enamel matrix protein, wherein the crude extract of enamel matrix protein is an acetic acid solution of enamel matrix protein; S2. adding trichloroacetic acid solution to the crude enamel matrix protein extract, shaking, incubating, so that the precipitate is fully separated; That In the experiment, the concentration of trichloroacetic acid in the crude extract of enamel matrix protein was 1.35% ≤ c ≤ 1.40%; S3. The incubated mixture was centrifuged and the supernatant was collected; S4. Continue to add trichloroacetic acid solution to the collected supernatant, control the concentration of trichloroacetic acid in the solution to 1.40%≤c≤1.7%, and then shake and incubate; after the precipitate is fully precipitated, centrifuge, collect the precipitate, and then resuspend it with ethanol to remove residual trichloroacetic acid, then centrifuge to remove ethanol, stand and dry, and then obtain amelogenin P148.

2. A process for extracting high-purity amelogenin according to claim 1, It is characterized in that In step S2, The trichloroacetic acid solution is added dropwise into the crude enamel matrix protein extract.

3. The extraction process of high-purity amelogenin according to claim 1, It is characterized in that In step S2, The incubation time is 1 to 5 minutes.

4. The extraction process of high-purity amelogenin according to claim 1, It is characterized in that In step S3, The centrifugal force of the centrifugation is 3000-5000g, and the centrifugal time is 3-10min.

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