CN115490750A - A kind of polypeptide synthesis, purification method and its application - Google Patents

Abstract

The present invention proposes a polypeptide synthesis and purification method and its application, specifically including the application of the compound represented by formula I or its salt in protein purification. According to the embodiment of the present invention, the compound represented by formula I can be used as a precipitation tag, and the protein can be effectively purified by using its precipitation or dissolution state in different polar reagents. The purification process is simple, effective and environmentally friendly.

Description

A kind of polypeptide synthesis, purification method and its application

technical field

The present invention relates to the field of biotechnology, in particular, the present invention relates to a method for synthesizing and purifying a polypeptide and its application.

Background technique

Nowadays, peptides play an increasingly important role in the development of drugs, biomaterials, diagnostic reagents and cosmetics. The acquisition of peptides mainly relies on solid-phase synthesis techniques. During the solid-phase synthesis of peptides, amino acids need to be continuously condensed on the solid-phase support. Due to the incomplete reaction in the condensation process, by-products will be produced, so an additional step of separation and purification is required. High-performance liquid chromatography (HPLC) was used for the separation of peptides after solid-phase synthesis due to its high separation efficiency. But this commonly used separation strategy also has disadvantages. First of all, a single liquid chromatography cannot perform parallel separation, and if you want to scale up the production level, you need a lot of equipment investment. At the same time, as the length of the polypeptide chain increases, impurities increase. If some impurities have the same retention time as the product, such impurities are also difficult to purify. Some hydrophobic peptides are also difficult to purify by HPLC. Due to their hydrophobic properties, these peptides will adhere to the chromatographic column, resulting in increased system pressure and separation and purification cannot be achieved. Finally, the entire HPLC separation and purification process consumes a large amount of chromatographically pure organic reagents, which is costly and environmentally unfriendly.

Therefore, it is still necessary to develop new peptide synthesis strategies on the basis of solid-phase synthesis to avoid separation using HPLC, which has important economic and environmental values.

Contents of the invention

The purpose of the present invention is to solve at least one of the above technical problems.

In the first aspect of the present invention, the present invention proposes the use of a compound represented by formula I or a salt thereof in purifying proteins,

According to an embodiment of the present invention, the compound represented by formula I or its salt can be dissolved in a less polar solvent, and can precipitate in a highly polar solvent environment, and its aldehyde group can react with the exposed amino group at the N-terminal of the polypeptide , compared with the method of HPLC purification and separation of polypeptides, it is more effective for the separation and purification of longer polypeptide chains and hydrophobic peptides, and the purification process is simpler, which reduces costs and is more environmentally friendly.

In some embodiments of the present invention, the protein is obtained through solid-phase synthesis.

In a second aspect of the present invention, the present invention proposes a method for purifying a protein. According to an embodiment of the present invention, the method includes: linking the protein sample to be purified with the compound represented by formula I or its salt; subjecting the linking product to precipitation; and subjecting the precipitated product to reconstitution to obtain the said protein;

According to an embodiment of the present invention, the compound represented by formula I or its salt can be dissolved in a less polar solvent, and can precipitate in a highly polar solvent environment, and its aldehyde group can react with the exposed amino group at the N-terminal of the polypeptide Therefore, compared with the method of purifying and separating polypeptides by HPLC, the method for purifying proteins described in this application is more effective for the separation and purification of longer polypeptide chains and hydrophobic peptides, and the purification process is simpler, reduces costs, and is more environmentally friendly.

According to an embodiment of the present invention, the method for purifying a protein may further include at least one of the following additional technical features:

According to an embodiment of the present invention, the mass ratio of the protein sample to be purified to the compound represented by formula I or its salt is 1:1-3:2. When the mass ratio of the protein sample to be purified to the compound represented by formula I or its salt is 1:1-3:2, the compound or its salt can fully contact and connect with the protein.

According to an embodiment of the present invention, the precipitation treatment is performed in a highly polar solvent.

According to an embodiment of the present invention, the reconstitution treatment is performed in a low-polarity reagent.

According to an embodiment of the present invention, the mass ratio of the protein sample to be purified to the compound represented by formula I or its salt is 57:46. When the mass ratio of the protein sample to be purified to the compound represented by formula I or its salt is 57:46, the compound or its salt can more fully contact and connect with the protein.

According to an embodiment of the present invention, in the linking process, -NH ₂ at the N-terminal of the protein undergoes a linking reaction with the aldehyde group of the compound.

According to an embodiment of the present invention, the ligation treatment is performed under the condition of pH<7.0. The ligation treatment is carried out under weak acid conditions, and the choice of the acid is not particularly limited, as long as the pH can be adjusted.

According to an embodiment of the present invention, the connection treatment is performed in a THF-DMF mixed solution.

According to an embodiment of the present invention, the mass ratio of THF and DMF in the THF-DMF mixture is 2:1˜3:1.

According to an embodiment of the present invention, the THF-DMF mixture further contains acetic acid.

According to an embodiment of the present invention, the volume ratio of acetic acid in the THF-DMF mixed solution is 0.1%.

According to an embodiment of the present invention, the connection treatment is carried out at 50° C. to 60° C. for 4.5 to 5.5 hours.

According to an embodiment of the present invention, the mass volume ratio of the ligated product to the highly polar solvent is 29 (mg): 2 (mL)-15 (mg): 1 (mL).

According to an embodiment of the present invention, the mass volume ratio of the ligated product to the highly polar solvent is 76 (mg): 5 (mL).

According to an embodiment of the present invention, the polarity of the highly polar solvent is not lower than 5.5.

According to an embodiment of the present invention, the highly polar reagent includes at least one selected from acetonitrile, acetic acid and aniline.

According to an embodiment of the present invention, after the precipitation treatment and before the reconstitution treatment, it further includes performing a first centrifugation treatment on the precipitation treatment product.

According to an embodiment of the present invention, the first centrifugation treatment is performed at 3000rpm-4000rpm for 2-4min, and centrifuged 1-3 times.

According to an embodiment of the present invention, the first centrifugation treatment is carried out at 3500 rpm for 3 minutes and centrifuged twice.

According to an embodiment of the present invention, the mass-to-volume ratio of the precipitation treatment product to the low-polarity solvent is 45 (mg): 1 (mL) to 40 (mg): 1 (mL). Under this ratio, the precipitation treatment product can be fully contacted with the low-polarity solvent, so as to dissolve and disconnect the linkage between the protein in the precipitation treatment product and the compound represented by formula I or its salt .

According to an embodiment of the present invention, the mass-to-volume ratio of the precipitation treatment product to the low-polarity solvent or solvent is 206 (mg): 5 (mL).

According to an embodiment of the present invention, the polarity of the low polarity solvent is lower than 3.5.

According to an embodiment of the present invention, the low-polarity solvent is selected from diethyl ether and/or methyl tert-butyl ether.

According to an embodiment of the present invention, the reconstitution treatment is performed in a low-polarity reagent and TFA.

According to an embodiment of the present invention, the mass-to-volume ratio of the low-polarity reagent to TFA is 6 (mg): 1 (mL) to 4 (mg): 1 (mL).

According to an embodiment of the present invention, the mass-to-volume ratio of the low-polarity reagent to TFA is 5 (mg): 1 (mL).

According to an embodiment of the present invention, the reconstitution treatment is performed in a low-polarity reagent, TFA, H ₂ O and Tips.

According to an embodiment of the present invention, the mass volume ratio of the low polarity reagent, TFA, H ₂ O and Tips is 40 (mL): 2 (mL): 2000 (mg): 1 (mL).

According to an embodiment of the present invention, the Tips include triisopropylsilane. Those skilled in the art can understand that the Tips are not particularly limited.

According to an embodiment of the present invention, the protein includes a protein obtained by a solid-phase polypeptide synthesis method.

According to an embodiment of the present invention, the redissolution treatment is to carry out shaking reaction at 24°C-28°C for 2-3 hours.

According to an embodiment of the present invention, the dissolution treatment is a shaking reaction at 26° C. for 2.5 hours.

According to an embodiment of the present invention, the method further includes re-precipitation treatment.

According to an embodiment of the present invention, the reprecipitation treatment is performed by contacting the reconstitution treatment product with a precipitation reagent to obtain the protein. During the reprecipitation process, the compound represented by formula I or its salt in the redissolution treatment product will form a precipitate again, and the protein is still dissolved in the precipitation reagent, thereby separating and purifying the two.

According to an embodiment of the present invention, after the re-dissolution treatment and before the re-precipitation treatment, it further includes concentrating the re-dissolution treatment product.

According to an embodiment of the present invention, the volume of the concentrated treatment product is 2-5 mL.

According to an embodiment of the present invention, during the reprecipitation treatment, the mass-to-volume ratio of the sample to be purified to the precipitation reagent is 8 (mg): 1 (mL) to 10 (mg): 1 (mL).

According to an embodiment of the present invention, during the reprecipitation treatment, the mass-to-volume ratio of the sample to be purified to the precipitation reagent is 228 (mg): 5 (mL).

According to an embodiment of the present invention, it further includes performing a second centrifugation treatment on the reprecipitation treatment product, so as to obtain the protein.

According to an embodiment of the present invention, the second centrifugation treatment is performed at 3000-4000 rpm for 2-4 minutes.

According to an embodiment of the present invention, the second centrifugation treatment is performed at 3500 rpm for 3 minutes.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows a strategy diagram of using the Fmoc method to synthesize proteins in solid phase and to precipitate homozygous tags to obtain proteins according to the embodiment of the present invention;

Fig. 2 has shown the HPLC detection result figure of the protein that the Fmoc method solid-phase synthesis protein of the embodiment of the present invention is not purified;

Fig. 3 has shown the HPLC detection result figure of the Fmoc method solid-phase synthesis protein of the embodiment of the present invention after adopting described label to purify protein;

Fig. 4 shows the results of mass spectrometry detection of the protein obtained after purification of the protein synthesized by the Fmoc method in solid phase using the tag according to the embodiment of the present invention.

detailed description

Embodiments of the invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the figures are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

In addition, the terms "first" and "second" are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

Neither the endpoints nor any values of the ranges disclosed herein are limited to such precise ranges or values, and these ranges or values are understood to include values approaching these ranges or values. For numerical ranges, between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, these values Ranges should be considered as specifically disclosed herein.

For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Abbreviations for amino acid residues are the standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

The abbreviations used in the present invention are contrasted as follows:

Fmoc: fluorenylmethoxycarbonyl;

HPLC: high performance liquid chromatography;

HCTU: Tetramethyluronium hexafluorophosphate;

DIEA: N,N-Diisopropylethylamine;

Oxyma: ethyl 2-oxime cyanoacetate;

DCM: dichloromethane;

DMF: N,N-dimethylformamide;

HOBt: 1-hydroxybenzotriazole;

TFA: trifluoroacetic acid;

Tips: Triisopropylsilane.

The present invention mainly relies on the property that the compound represented by formula I or its salt (precipitation tag) can be dissolved in a less polar solvent and precipitate in a highly polar solvent environment. Combining the compound or its salt with classic Fmoc solid-phase synthesis, the aldehyde group on the precipitation label and the exposed amino group at the N-terminal of the polypeptide undergo a schiff base intermediate under weakly acidic conditions, thereby separating the polypeptide in acetonitrile/water polarity Precipitation under these conditions afforded pure fully protected intermediates, which were then worked up to yield lyophilized pure peptides without HPLC purification. In addition, the inventors conducted a large number of experimental screening and optimization of various parameters and reagents in the purification method, so that the method described in this application can purify the protein more efficiently, and the strategy described in this application was used to synthesize, separate and purify the protein or peptide strategy as shown in Figure 1:

1) Using the classic Fmoc solid-phase synthesis strategy, after each amino acid condensation is completed, acetic anhydride is added to block the unreacted amino groups. Until the solid-phase synthesis of the peptide is completed.

2) Cutting the polypeptide obtained in step 1) for full protection.

3) React the fully protected polypeptide with the precipitation tag, and add glacial ether to precipitate the polypeptide after the reaction is completed.

4) Collect the precipitate for cleavage, remove the precipitate tag and side chain protection, and use ether precipitation to initially obtain pure peptide.

5) The precipitate is dissolved in acetonitrile/water, then reprecipitated, filtered to remove the precipitated label, and the filtrate is freeze-dried to obtain a freeze-dried polypeptide.

The method of protein purification using the precipitation tag does not require HPLC to separate and purify polypeptides, and can realize green synthesis of polypeptides, greatly saving costs such as HPLC equipment, reagents, and time.

The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are only illustrative and do not limit the present invention in any way. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

The synthesis of embodiment 1 polypeptide

This example is used to synthesize the target polypeptide. Weigh 1100mg of Wang resin (0.4mmol) with a degree of substitution of 0.36mmol/g into a polypeptide synthesis tube, add 5mL of DMF and 5mL of DCM, and place at room temperature for 30min to swell the resin. Weigh 619 mg (1.6 mmol) of Fmoc-Phe-OH (phenylalanine), put it into a 50 mL centrifuge tube, add 10 mL of DMF to dissolve, and then add 528 μL (3.2 mmol) of DIEA. Transfer the above mixed solution to a peptide synthesis tube, shake and react at 33°C overnight, drain and wash the resin. Add 10mL methanol/DMF (1:20) mixed solution to the resin, shake and react at 33°C for 10min, drain and wash the resin. Add 10 mL of 20% piperidine solution (0.1 M oxyma) to the peptide synthesis tube to remove Fmoc, and react twice at 33° C. for 5 min+10 min. Weigh 565mg (1.6mmol) of Fmoc-Ile-OH (isoleucine), 628mg (1.52mmol) of condensing agent HCTU, put them into a 50mL centrifuge tube, use 10mL DMF to dissolve, then add 528μL (3.2mmol ) of the DIEA. Transfer the above mixed solution to a peptide synthesis tube, shake and react at 33°C for 1 h, drain and wash the resin after the reaction is complete. Add 10mL of acetic anhydride: DIEA: DMF (1:1:8) mixed solution to the resin, block unreacted amino acid, shake and react at 33°C for 15min, react twice in total, drain and wash the resin after completion. According to the polypeptide sequence, the remaining amino acids undergo amino acid condensation, amino acid blocking and Fmoc deprotection in sequence. Use 4 equiv of Fmoc to protect amino acids, 3.8 equiv of HCTU and 8 equiv of DIEA for 1 h.

10 mL of 1% TFA/DCM solution was added to the resin to carry out full-protection cleavage of the polypeptide, and after shaking at 33° C. for 1 h, the filtrate was collected by filtration. Use a rotary evaporator to remove excess DCM from the filtrate, add an appropriate amount of glacial ether to precipitate, and centrifuge at 3500 rpm to collect the precipitate. Repeat the ether wash process once. The precipitate was collected and dried to obtain crude peptide.

The purification of embodiment 2 polypeptide

Weigh 10 mg of the crude peptide obtained in Example 1, and directly add 1 mL of cleavage reagent to deprotect the fully protected polypeptide. The reaction was shaken at 26°C for 2.5h. After the reaction was completed, glacial ether was added, and the precipitate was collected after centrifugation at 3500 rpm. After the precipitate was dissolved in 5 mL of acetonitrile/water, it was analyzed by HPLC as a comparison of the purity of the peptide before using the precipitation label. The specific experimental results are shown in Figure 2, in which there are obvious impurity absorption peaks on the side of the main peak absorption position.

Weigh 228 mg of the crude peptide obtained in Example 1 and 2 equiv precipitation tag (184 mg, obtained by the inventor himself) into a 50 mL EP tube, add 10 mL THF:DMF (7:3) to dissolve, and then add 100 μL of acetic acid. React at 55°C for 5 hours to attach the peptide to the precipitation tag. Take out the 50mL EP tube, add 15mL of acetonitrile, shake the EP tube properly and observe the precipitation, then put the EP tube into the centrifuge at 3500rpm, and centrifuge for 3min. After the centrifugation was completed, the precipitate was collected, repeated twice, air-dried at room temperature, and mashed after drying. After adding 10 mL of TFA/H ₂ O/phenol/Tips (10 mL/500 μL/500 mg/250 μL) to the above precipitate, the reaction was shaken at 26° C. for 2.5 h. Using nitrogen sparging at room temperature, the solution was concentrated to less than 5 mL. Add 25mL of acetonitrile to the concentrated solution, oscillate to see the precipitation and centrifuge at 3500rpm for 3min; collect the supernatant and use HPLC for analysis to identify the change in the purity of the polypeptide after using the precipitation tag, and perform mass spectrometry detection on the protein obtained by purifying the tag. The experimental results are shown in Figure 3 and Figure 4. The results show that after the purification of the tag-treated polypeptide, the impurity absorption peaks on the side of the original main peak have been removed, and the overall chromatographic yield has reached more than 95%. Moreover, the molecular weight of the synthetic polypeptide was also correctly characterized by ESI-MS, which was 1015.6 Da. After being lyophilized by a lyophilizer, it weighed 54 mg, and the yield was 36%.

In addition, the terms "first" and "second" are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the described specific features, structures, materials or characteristics may be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples and features of different embodiments or examples described in this specification without conflicting with each other.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and those skilled in the art can make the above-mentioned The embodiments are subject to changes, modifications, substitutions and variations.

Claims (12)

1. The purposes of the compound shown in formula I or its salt in purifying protein,

2. A method for purifying proteins, comprising: Linking the protein sample to be purified with the compound represented by formula I or its salt; subjecting the ligation product to precipitation; and reconstitute the precipitated product to obtain the protein;

3. The method according to claim 2, wherein the mass ratio of the protein sample to be purified to the compound represented by formula I or its salt is 1:1-3:2. 4. The method according to claim 2, characterized in that, the precipitation treatment is carried out in a highly polar solvent; Optionally, the reconstitution treatment is performed in a low-polarity reagent. 5. The method according to claim 2, wherein the mass ratio of the protein sample to be purified to the compound represented by formula I or its salt is 57:46; Optionally, in the connection treatment, the _-NH2 at the N-terminal of the protein reacts with the aldehyde group of the compound; Optionally, the ligation treatment is carried out at pH<7.0; Optionally, the connection treatment is carried out in THF-DMF mixed solution; Optionally, the mass ratio of THF and DMF in the THF-DMF mixture is 2:1-3:1; Optionally, the THF-DMF mixture further contains acetic acid; Optionally, the volume ratio of acetic acid in the THF-DMF mixed solution is 0.1%; Optionally, the connection treatment is carried out at 50°C-60°C for 4.5-5.5h. 6. The method according to claim 4, characterized in that the mass volume ratio of the ligation product to the highly polar solvent is 29 (mg): 2 (mL) to 15 (mg): 1 (mL) ; Optionally, the mass volume ratio of the ligation product to the highly polar solvent is 76 (mg): 5 (mL); Optionally, the polarity of the highly polar solvent is not lower than 5.5; Optionally, the highly polar reagent includes at least one selected from acetonitrile, acetic acid and aniline. 7. The method according to claim 2, characterized in that, after the precipitation treatment and before the reconstitution treatment, further comprising subjecting the precipitation treatment product to a first centrifugation treatment; Optionally, the first centrifugation treatment is carried out at 3000rpm-4000rpm for 2-4min, centrifuged 1-3 times; Optionally, the first centrifugation treatment is carried out at 3500 rpm for 3 minutes and centrifuged twice. 8. The method according to claim 4, characterized in that, the mass volume ratio of the precipitation treatment product to the low polarity solvent is 45 (mg): 1 (mL) ~ 40 (mg): 1 (mL ); Optionally, the mass volume ratio of the precipitation treatment product to the low polarity solvent or the solvent is 206 (mg): 5 (mL); Optionally, the polarity of the low polarity solvent is lower than 3.5; Optionally, the low-polarity solvent is selected from diethyl ether and/or methyl tert-butyl ether. 9. The method according to claim 2, characterized in that, the reconstitution treatment is carried out in low polarity reagent and TFA; Optionally, the mass volume ratio of the low polarity reagent to TFA is 6 (mg): 1 (mL) ~ 4 (mg): 1 (mL); Optionally, the mass volume ratio of the low polarity reagent to TFA is 5 (mg): 1 (mL); Optionally, the reconstitution treatment is carried out in low polar reagents, TFA, H ₂ O and Tips; Optionally, the mass volume ratio of the low polarity reagent, TFA, H ₂ O and Tips is 40 (mL): 2 (mL): 2000 (mg): 1 (mL); Optionally, the Tips include triisopropylsilane; Optionally, the protein comprises a protein obtained using solid phase polypeptide synthesis. 10. The method according to claim 9, characterized in that, the reconstitution treatment is performed at 24°C-28°C for 2-3 hours of shaking reaction; Optionally, the dissolution treatment is a shaking reaction at 26° C. for 2.5 hours. 11. The method according to claim 2, further comprising re-precipitation treatment; Optionally, the reprecipitation treatment is carried out as follows: contacting the reconstituted product with a precipitation reagent to obtain the protein; Optionally, after the re-dissolution treatment and before the re-precipitation treatment, further comprising concentrating the re-dissolution treatment product; Preferably, the volume of the concentrated treatment product is 2-5 mL; Optionally, during the reprecipitation process, the mass volume ratio of the sample to be purified to the precipitation reagent is 8 (mg): 1 (mL) to 10 (mg): 1 (mL); Optionally, during the reprecipitation treatment, the mass volume ratio of the sample to be purified to the precipitation reagent is 228 (mg): 5 (mL). 12. The method according to claim 11, further comprising performing a second centrifugal treatment on the reprecipitation product to obtain the protein; Optionally, the second centrifugation treatment is carried out at 3000-4000 rpm for 2-4 minutes; Optionally, the second centrifugation treatment is performed at 3500 rpm for 3 minutes.

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