CN116162124B - Preparation method of continuous glutamine polypeptide - Google Patents
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Abstract
The invention discloses a preparation method of continuous glutamine polypeptide, which mainly solves the technical problems that the solubility of polypeptide synthesized by solid phase is poorer than that of single amino acid and the glutamine deamidation of nitrogen terminal. The method comprises the following steps: 1) Solid-phase synthesized continuous glutamine polypeptide, wherein glu at the N end is protected by fmoc to form fmoc-continuous glutamine polypeptide; 2) Fully dissolving with hexafluoroisopropanol, and measuring the molecular weight of the crude liquid; 3) Determining a first step chromatographic condition; 4) Deprotection of the collected peptide solution with piperidine fmoc to give a continuous glutamine polypeptide solution and determination of molecular weight using ESI-MS; 5) Replacing the original reagent with flash medium-pressure column for the deprotected peptide solution; 6) Determining the chromatographic conditions of the third step; 7) And (3) freezing and drying the obtained peptide solution by liquid nitrogen to obtain a white powdery finished product. The invention can obtain stable high-purity continuous glutamine polypeptide.
Description
Technical Field
The invention belongs to the technical field of polypeptide preparation, and particularly relates to a preparation method for obtaining stable high-purity continuous glutamine polypeptide. The invention is useful for preparing polypeptides by reverse phase HPLC in combination with electrospray mass spectrometry (ESI-MS).
Background
Glutamine, the name 2-amino-4-carbamoyl butyric acid, english Glutamine (Gln), abbreviated Q. L-glutamine is an amino acid encoded in protein synthesis, a mammalian non-essential amino acid, which can be converted from glucose in vivo. Glutamine can be used for treating gastric and duodenal ulcer, gastritis and gastric hyperacidity, and for improving brain function. Sealing and preserving at a ventilation place. White crystals or crystalline powder, soluble in water, insoluble in methanol, ethanol, ether, benzene, acetone, chloroform and ethyl acetate, odorless and slightly sweet. Unstable in neutral solution, can be easily decomposed into glutamine or propyl esterified into pyrrolecarboxyl alcohol in alcohol, alkali or hot water, has no odor, and has slight sweet taste.
Since a polypeptide formed by a reaction involving a plurality of continuous glutamine is too much in the case of continuously repeating synthesis of glutamine, beta folding is easily formed, and the solubility of the polypeptide synthesized in a solid phase becomes worse than that of a single amino acid. The greater the number of glutamine, the more hydrophobic the solubility of the entire peptide fragment becomes. In particular, glutamine at the nitrogen end of polypeptide is relatively active, and glutamine is easily hydrolyzed and deamidated to be glutamic acid in neutral aqueous solution, alkaline solution or under high temperature condition. The difficulty of preparing such polypeptides by reverse-phase high performance liquid chromatography is greatly increased due to the hydrophobicity and extreme instability of such polypeptides. In order to solve the problem of deamidation of glutamine at the nitrogen end, the invention provides a method for protecting glutamine at the nitrogen end, performing first-step rapid preparation, performing deprotection of the nitrogen end, and selecting proper storage solvent conditions and preparation conditions, so that a plurality of continuous glutamine can be effectively separated, and the stability of the whole polypeptide in the preparation process can be ensured.
Disclosure of Invention
The invention aims to provide a technical method suitable for preparing a stable high-purity continuous glutamine polypeptide, which mainly solves the technical problems that the solubility of the polypeptide synthesized by a solid phase is poorer than that of single amino acid and the glutamine deamidation at the nitrogen end.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a method of preparing a plurality of continuous glutamine polypeptides comprising the steps of:
1) Firstly, synthesizing a solid polypeptide crude product of fmoc-continuous glutamine polypeptide with fmoc protection at the N end by using a solid phase synthesis method;
2) Fully dissolving the fmoc-continuous glutamine polypeptide solid polypeptide crude product synthesized by a solid phase by using hexafluoroisopropanol, measuring the molecular weight of the solution by using an electrospray mass spectrometry (ESI-MS), and filtering to obtain fmoc-continuous glutamine polypeptide crude liquid as a sample solution;
3) The first step of rapid preparation, wherein a non-porous carrier column is used for preparing a chromatographic column, a hydrochloric acid aqueous solution with the mass percent concentration of 0.01% is used as an A phase, chromatographic pure acetonitrile is used as a B phase, the chromatographic column is washed clean by acetonitrile and then is put into a sample solution, the mass ratio of the linear preparation gradient acetonitrile concentration is set to be 8-50%, and a peptide solution with a target peak value is collected;
4) Performing fmoc deprotection on the peptide solution obtained by the rapid preparation in the first step by using hexahydropyridine with the mass percentage concentration of 15-20% to obtain a continuous glutamine linear polypeptide solution, determining the molecular weight of the solution by using electrospray mass spectrometry, and preserving at low temperature;
5) The second step of preparation, solution replacement, namely replacing the original reagent by using a flash medium-pressure column and using a hydrochloric acid aqueous solution with the mass percent concentration of 0.1% as a leaching agent;
6) The third step of accurate chromatographic preparation, namely, a reverse phase silica gel column with a stationary phase being tetraalkyl bonded silica gel, wherein 0.01 mass percent hydrochloric acid aqueous solution is used as phase A, chromatographic pure acetonitrile is used as phase B, the concentration mass ratio of linear preparation gradient acetonitrile is set to be 12-33%, and peptide solution with a target peak value is collected again;
7) And (3) performing liquid nitrogen freezing and drying on the peptide solution prepared by the third step of accurate chromatography to obtain a white powdery stable hydrochloride form finished product.
The preferable scheme is as follows: the dissolution temperature of the step 2) is 40 ℃.
The preferable scheme is as follows: the chromatographic column in the step 3) is 5-8um particles.
The preferable scheme is as follows: the preparation flow rate of the system in the step 3) is 5-10ml/min.
The preferable scheme is as follows: the chromatographic column in the step 3) is added with a column temperature box at 20 ℃.
The preferable scheme is as follows: the low-temperature preservation in the step 4) is 10 ℃.
The preferable scheme is as follows: the diameter of the flash medium-pressure column in the step 5) is 20-30mm, and a column temperature box is added at 10 ℃.
The preferable scheme is as follows: the leaching speed of the step 5) is 17-22ml/min, and the pH value is measured until the pH value is acidic.
The preferable scheme is as follows: the reversed phase silica gel column in the step 6) has the particle size of 10um and the aperture of 300A, and the column temperature is increased to 20 ℃.
The beneficial effects of the invention are as follows: aiming at the prior art of preparing a plurality of continuous glutamine polypeptides, the invention provides a process method suitable for preparing a plurality of continuous glutamine polypeptides, which uses an indirect synthesis target polypeptide mode to prepare a plurality of continuous glutamine polypeptide finished products in the form of white powdery stable hydrochloride through multi-step preparation and different chromatographic column types, mobile phase conditions and temperature conditions. The invention provides a method for preparing continuous glutamine polypeptide by utilizing reversed phase HPLC and electrospray mass spectrometry (ESI-MS) combined technology aiming at the polypeptide. And (3) performing rapid preparation on a plurality of continuous glutamine polypeptides with the nitrogen end subjected to fmoc protection by using a pore-free carrier column, performing fmoc deprotection on intermediate liquid after preparation, and replacing intermediate liquid reagents after deprotection, so that the plurality of continuous glutamine polypeptide solutions are in stable environmental conditions, and the final step is performed to accurately prepare the stable high-purity continuous glutamine polypeptides. The whole process research is carried out in a low-temperature environment, and different temperature settings can ensure the stability of the polypeptide according to different stage research processes, and delay the separation time of the polypeptide in a chromatographic column so as to achieve a better separation effect. The invention supplements the blank and technical defects of the prior preparation technology of continuous multiple glutamine polypeptides, and adds a new technology and a new research direction for the preparation and separation of continuous multiple glutamine polypeptides.
Drawings
FIG. 1 is a HPLC chart of a peptide solution obtained by the first step of the present invention.
FIG. 2 is a HPLC chart of the product of the present invention.
Detailed Description
Example 1
1. Solid phase synthesis: firstly, synthesizing a solid polypeptide crude product with the nitrogen terminal protected by fmoc by using a solid phase synthesis method. ([ Tang Hong, sun Lishu, chen Wuling. Fmoc solid phase Synthesis of JFT Process study [ J ]. Modern biomedical advances.2007, (01): 85-87. ]).
2. Sample treatment: and fully dissolving the synthesized fmoc solid polypeptide crude product with hexafluoroisopropanol at 40 ℃ (the dissolving concentration is about 50 mg/ml), filtering the fully dissolved liquid by using a 0.45um organic phase filter head, reserving filtrate as a sample solution for later use, and carrying out molecular weight correctness verification on the crude product liquid by using an electrospray mass spectrometer (ESI-MS).
3. First step of rapid preparation
The preparation conditions are as follows: chromatographic column: the non-porous carrier column is a preparative chromatographic column, and the diameter and the length of the column are as follows: 2cm x 20cm, 5um particles, column incubator 20 ℃. Mobile phase: phase A: proportioning hydrochloric acid aqueous solution with mass percentage concentration of 0.01%; and B phase: acetonitrile was chromatographically pure. Flow rate: 5ml/min. Detection wavelength: 210nm. Gradient: mass percent concentration of mobile phase B: 8-48%, and gradient treatment time is 2-10min. The sample injection amount is 100mg;
the rapid preparation process comprises the following steps: and (3) washing the chromatographic column with acetonitrile with the mass percentage concentration of more than 90%, and then loading the sample, wherein the loading amount is the sample solution. Linear gradient elution, collecting target peak with purity up to 80% (figure 1), and storing the collected peptide solution in 10 deg.c environment.
4. Deprotection treatment: and (3) proportioning the piperidine with the mass percentage concentration of 15% and the DMF solution with the mass percentage concentration of 100%, performing fmoc deprotection treatment on the peptide solution prepared rapidly in the first step to obtain a target polypeptide solution after deprotection, and storing the target polypeptide solution at 10 ℃ for standby, and performing deprotection by using an electrospray mass spectrometry (ESI-MS) again to verify the correctness of the molecular weight of the product liquid.
5. Second step of displacement preparation
The preparation conditions are as follows: chromatographic column: flash medium-pressure column, the diameter and the length of the column are: 2cm by 30cm, and a column incubator at 10 ℃. The medium pressure pump was configured to have a maximum flow rate of 30ml/min. Mobile phase: the hydrochloric acid aqueous solution with the mass percentage concentration of 0.1 percent is the eluent. Flow rate: 17ml/min;
the preparation process comprises the following steps: and (3) passing the target polypeptide solution after deprotection through a flash medium-pressure column, collecting the liquid after passing through the column, measuring the pH of the solution of the collected liquid, repeatedly passing through the flash column for a plurality of times until the pH of the collected sample solution is acidic, and keeping the temperature of the collected liquid at 10 ℃.
6. Third step of accurate preparation
The preparation conditions are as follows: chromatographic column: reverse phase silica gel column using stationary phase as tetraalkyl bond silica gel, particle diameter is 10um, aperture is 300A, column diameter and length are: 2cm by 20cm, and a column incubator at 20 ℃. Mobile phase: phase A: preparing hydrochloric acid aqueous solution with the mass percentage concentration ratio of 0.01%; and B phase: acetonitrile was chromatographically pure. Flow rate: 20ml/min. Detection wavelength: 215nm. Gradient: mass percent concentration of mobile phase B: 10-35%, and gradient treatment time is 2-25min;
the preparation process comprises the following steps: and (3) washing the chromatographic column with acetonitrile with the mass percentage concentration of more than 90%, and then loading the sample, wherein the loading amount is the sample solution. And (3) carrying out linear gradient elution, collecting a target peak, and preserving a target peak solution at 10 ℃.
7. Post-treatment: and (3) freezing the continuous multiple glutamine polypeptides obtained after accurate preparation by liquid nitrogen, and drying in vacuum to finally obtain white powdery hydrochloride-form polypeptide finished products, wherein the purity is more than 98.0%, and the yield can be more than 80.6% (shown in figure 2).
Example 2
1. Solid phase synthesis: firstly, synthesizing a solid polypeptide crude product with the nitrogen terminal protected by fmoc by using a solid phase synthesis method.
2. Sample treatment: and fully dissolving the synthesized fmoc solid polypeptide by using hexafluoroisopropanol at 40 ℃ (the dissolving concentration is about 50 mg/ml), filtering the fully dissolved liquid by using a 0.45um organic phase filter head, reserving filtrate for later use, and carrying out verification on the molecular weight correctness of the crude liquid by using electrospray mass spectrometry (ESI-MS).
3. First step of rapid preparation
The preparation conditions are as follows: chromatographic column: the non-porous carrier column is a preparative chromatographic column, and the diameter and the length of the column are as follows: 2cm x 25cm, 8um particles, column incubator 20 ℃. Mobile phase: phase A: proportioning hydrochloric acid aqueous solution with mass percentage concentration of 0.01%; and B phase: acetonitrile was chromatographically pure. Flow rate: 7ml/min. Detection wavelength: 210nm. Gradient: mass percent concentration of mobile phase B: 8-50%, and gradient treatment time is 2-10min. The sample injection amount is 200mg;
the rapid preparation process comprises the following steps: and (3) washing the chromatographic column with acetonitrile with the mass percentage concentration of more than 90%, and then loading the sample, wherein the loading amount is the sample solution. And (3) carrying out linear gradient elution, collecting target peaks, wherein the purity of the target peaks can reach more than 80%, and storing the collected peptide solution in a 10-DEG environment for standby.
4. Deprotection treatment: and (3) proportioning the piperidine with the mass percentage concentration of 18% and the DMF solution with the mass percentage concentration of 100%, performing fmoc deprotection treatment on the peptide solution prepared rapidly in the first step to obtain a target polypeptide solution after deprotection, and storing the target polypeptide solution at 10 ℃ for standby, and performing deprotection by using an electrospray mass spectrometry (ESI-MS) again to verify the correctness of the molecular weight of the product liquid.
5. Second step of displacement preparation
The preparation conditions are as follows: chromatographic column: flash medium-pressure column, the diameter and the length of the column are: 2cm by 30cm, and a column incubator at 10 ℃. The medium pressure pump was configured to have a maximum flow rate of 30ml/min. Mobile phase: the hydrochloric acid aqueous solution with the mass percentage concentration of 0.1 percent is the eluent. Flow rate: 19ml/min;
the preparation process comprises the following steps: and (3) passing the target polypeptide solution after deprotection through a flash medium-pressure column, collecting the liquid after passing through the column, measuring the pH of the solution of the collected liquid, repeatedly passing through the flash column for a plurality of times until the pH of the collected sample solution is acidic, and keeping the temperature of the collected liquid at 10 ℃.
6. Third step of accurate preparation
The preparation conditions are as follows: chromatographic column: reverse phase silica gel column using stationary phase as tetraalkyl bond silica gel, particle diameter is 10um, aperture is 300A, column diameter and length are: 2cm by 25cm, and a column incubator at 20 ℃. Mobile phase: phase A: proportioning hydrochloric acid aqueous solution with mass percentage concentration of 0.01%; and B phase: acetonitrile was chromatographically pure. Flow rate: 22ml/min. Detection wavelength: 215nm. Gradient: mass percent concentration of mobile phase B: 12-35%, and gradient treatment time is 2-25min;
the preparation process comprises the following steps: and (3) washing the chromatographic column with acetonitrile with the mass percentage concentration of more than 90%, and then loading the sample, wherein the loading amount is the sample solution. And (3) carrying out linear gradient elution, collecting a target peak, and preserving a target peak solution at 10 ℃.
7. Post-treatment: and (3) freezing the continuous glutamine polypeptides obtained after the precise preparation by liquid nitrogen, and drying in vacuum to finally obtain white powdery hydrochloride-form polypeptide finished products, wherein the purity is more than 98.0%, and the yield can be more than 81.3%.
Example 3
1. Solid phase synthesis: firstly, synthesizing a solid polypeptide crude product with the nitrogen terminal protected by fmoc by using a solid phase synthesis method.
2. Sample treatment: and fully dissolving the synthesized fmoc solid polypeptide by using hexafluoroisopropanol at 40 ℃ (the dissolving concentration is about 50 mg/ml), filtering the fully dissolved liquid by using a 0.45um organic phase filter head, reserving filtrate for later use, and carrying out verification on the molecular weight correctness of the crude liquid by using electrospray mass spectrometry (ESI-MS).
3. First step of rapid preparation
The preparation conditions are as follows: chromatographic column: the non-porous carrier column is a preparative chromatographic column, and the diameter and the length of the column are as follows: 3cm by 20cm, 5um particles, column incubator 20 ℃. Mobile phase: phase A: proportioning hydrochloric acid aqueous solution with mass percentage concentration of 0.01%; and B phase: acetonitrile was chromatographically pure. Flow rate: 9ml/min. Detection wavelength: 210nm. Gradient: mass percent concentration of mobile phase B: 10-48%, and gradient treatment time is 2-10min. The sample injection amount is 500mg;
the rapid preparation process comprises the following steps: and (3) washing the chromatographic column with acetonitrile with the mass percentage concentration of more than 90%, and then loading the sample, wherein the loading amount is the sample solution. And (3) carrying out linear gradient elution, collecting target peaks, wherein the purity of the target peaks can reach more than 80%, and storing the collected peptide solution in an environment of 10 ℃ for standby.
4. Deprotection treatment: and (3) proportioning the piperidine with the mass percentage concentration of 20% and the DMF solution with the mass percentage concentration of 100%, performing fmoc deprotection treatment on the peptide solution prepared rapidly in the first step to obtain a target polypeptide solution after deprotection, storing the target polypeptide solution at 10 ℃ for standby, and performing deprotection by using an electrospray mass spectrometry (ESI-MS) again to verify the correctness of the molecular weight of the product liquid.
5. Second step of displacement preparation
The preparation conditions are as follows: chromatographic column: flash medium-pressure column, the diameter and the length of the column are: 3cm by 30cm, and a column incubator at 10 ℃. The medium pressure pump was configured to have a maximum flow rate of 30ml/min. Mobile phase: the hydrochloric acid aqueous solution with the mass percentage concentration of 0.1 percent is the eluent. Flow rate: 20ml/min;
the preparation process comprises the following steps: and (3) passing the target polypeptide solution after deprotection through a flash medium-pressure column, collecting the liquid after passing through the column, measuring the pH of the solution of the collected liquid, repeatedly passing through the flash column for a plurality of times until the pH of the collected sample solution is acidic, and keeping the temperature of the collected liquid at 10 ℃.
6. Third step of accurate preparation
The preparation conditions are as follows: chromatographic column: reverse phase silica gel column using stationary phase as tetraalkyl bond silica gel, particle diameter is 10um, aperture is 300A, column diameter and length are: 3cm by 20cm, and a column incubator at 20 ℃. Mobile phase: phase A: proportioning hydrochloric acid aqueous solution with mass percentage concentration of 0.01%; and B phase: acetonitrile was chromatographically pure. Flow rate: 25ml/min. Detection wavelength: 215nm. Gradient: mass percent concentration of mobile phase B: 11-34%, and gradient treatment time is 2-25min;
the preparation process comprises the following steps: and (3) washing the chromatographic column with acetonitrile with the mass percentage concentration of more than 90%, and then loading the sample, wherein the loading amount is the sample solution. And (3) carrying out linear gradient elution, collecting a target peak, and preserving a target peak solution at 10 ℃.
7. Post-treatment: and (3) freezing the continuous glutamine polypeptides obtained after the precise preparation by liquid nitrogen, and drying in vacuum to finally obtain white powdery hydrochloride-form polypeptide finished products, wherein the purity is more than 98.0%, and the yield can be more than 82.4%.
Example 4
1. Solid phase synthesis: firstly, synthesizing a solid polypeptide crude product with the nitrogen terminal protected by fmoc by using a solid phase synthesis method.
2. Sample treatment: and fully dissolving the synthesized fmoc solid polypeptide by using hexafluoroisopropanol at 40 ℃ (the dissolving concentration is about 50 mg/ml), filtering the fully dissolved liquid by using a 0.45um organic phase filter head, reserving filtrate for later use, and carrying out verification on the molecular weight correctness of the crude liquid by using electrospray mass spectrometry (ESI-MS).
3. First step of rapid preparation
The preparation conditions are as follows: chromatographic column: the non-porous carrier column is a preparative chromatographic column, and the diameter and the length of the column are as follows: 3cm x 25cm, 8um particles, column incubator 20 ℃. Mobile phase: phase A: proportioning hydrochloric acid aqueous solution with mass percentage concentration of 0.01%; and B phase: acetonitrile was chromatographically pure. Flow rate: 10ml/min. Detection wavelength: 210nm. Gradient: mass percent concentration of mobile phase B: 10-50%, and gradient treatment time is 2-10min. The sample injection amount is 500mg;
the rapid preparation process comprises the following steps: and (3) washing the chromatographic column with acetonitrile with the mass percentage concentration of more than 90%, and then loading the sample, wherein the loading amount is the sample solution. And (3) carrying out linear gradient elution, collecting target peaks, wherein the purity of the target peaks can reach more than 80%, and storing the collected peptide solution in an environment of 10 ℃ for standby.
4. Deprotection treatment: and (3) proportioning the piperidine with the mass percentage concentration of 20% and the DMF solution with the mass percentage concentration of 100%, performing fmoc deprotection treatment on the peptide solution prepared rapidly in the first step to obtain a target polypeptide solution after deprotection, storing the target polypeptide solution at 10 ℃ for standby, and performing deprotection by using an electrospray mass spectrometry (ESI-MS) again to verify the correctness of the molecular weight of the product liquid.
5. Second step of displacement preparation
The preparation conditions are as follows: chromatographic column: flash medium-pressure column, the diameter and the length of the column are: 3cm by 30cm, and a column incubator at 10 ℃. The medium pressure pump was configured to have a maximum flow rate of 30ml/min. Mobile phase: the hydrochloric acid aqueous solution with the mass percentage concentration of 0.1 percent is the eluent. Flow rate: 22ml/min;
the preparation process comprises the following steps: and (3) passing the target polypeptide solution after deprotection through a flash medium-pressure column, collecting the liquid after passing through the column, measuring the pH of the solution of the collected liquid, repeatedly passing through the flash column for a plurality of times until the pH of the collected sample solution is acidic, and keeping the temperature of the collected liquid at 10 ℃.
6. Third step of accurate preparation
The preparation conditions are as follows: chromatographic column: reverse phase silica gel column using stationary phase as tetraalkyl bond silica gel, particle diameter is 10um, aperture is 300A, column diameter and length are: 3cm by 25cm, and 20℃in a column incubator. Mobile phase: phase A: proportioning hydrochloric acid aqueous solution with mass percentage concentration of 0.01%; and B phase: acetonitrile was chromatographically pure. Flow rate: 25ml/min. Detection wavelength: 215nm. Gradient: mass percent concentration of mobile phase B: 12-33%, and gradient treatment time is 2-25min;
the preparation process comprises the following steps: and (3) washing the chromatographic column with acetonitrile with the mass percentage concentration of more than 90%, and then loading the sample, wherein the loading amount is the sample solution. And (3) carrying out linear gradient elution, collecting a target peak, and preserving a target peak solution at 10 ℃.
7. Post-treatment: and (3) freezing the continuous glutamine polypeptides obtained after the precise preparation by liquid nitrogen, and drying in vacuum to finally obtain white powdery hydrochloride-form polypeptide finished products, wherein the purity is more than 98.0%, and the yield can be more than 83.7%.
Claims (7)
1. A method for producing a continuous glutamine polypeptide, characterized by: the method comprises the following steps:
1) Firstly, synthesizing a solid polypeptide crude product of fmoc-continuous glutamine polypeptide with fmoc protection at the N end by using a solid phase synthesis method;
2) Fully dissolving the fmoc-continuous glutamine polypeptide solid polypeptide crude product synthesized by a solid phase by hexafluoroisopropanol, measuring the molecular weight of the solution by electrospray mass spectrometry, and filtering to obtain fmoc-continuous glutamine polypeptide crude liquid as a sample solution;
3) The first step of rapid preparation, wherein a non-porous carrier column is used for preparing a chromatographic column, a hydrochloric acid aqueous solution with the mass percent concentration of 0.01% is used as an A phase, chromatographic pure acetonitrile is used as a B phase, the chromatographic column is washed clean by acetonitrile and then is put into a sample solution, the mass ratio of the linear preparation gradient acetonitrile concentration is set to be 8-50%, and a peptide solution with a target peak value is collected;
4) Performing fmoc deprotection on the peptide solution obtained by the rapid preparation in the first step by using hexahydropyridine with the mass percentage concentration of 15-20% to obtain a continuous glutamine linear polypeptide solution, determining the molecular weight of the solution by using electrospray mass spectrometry, and preserving at low temperature;
5) The second step of preparation, solution replacement, namely replacing the original reagent by using a flash medium-pressure column and using a hydrochloric acid aqueous solution with the mass percent concentration of 0.1% as a leaching agent;
6) The third step of accurate chromatographic preparation, namely, a reverse phase silica gel column with a stationary phase being tetraalkyl bonded silica gel, wherein 0.01 mass percent hydrochloric acid aqueous solution is used as phase A, chromatographic pure acetonitrile is used as phase B, the concentration mass ratio of linear preparation gradient acetonitrile is set to be 12-33%, and peptide solution with a target peak value is collected again;
7) And (3) performing liquid nitrogen freezing and drying on the peptide solution prepared by the third step of accurate chromatography to obtain a white powdery stable hydrochloride form finished product.
2. The method for producing a continuous glutamine polypeptide as defined in claim 1, wherein: the dissolution temperature of the step 2) is 40 ℃.
3. The method for producing a continuous glutamine polypeptide as defined in claim 1, wherein: the step 3) is carried out by adding 5-8um particles into a non-porous chromatographic column, preparing the mixture at flow rate of 5-10ml/min, and adding into a column temperature box at 20 ℃.
4. The method for producing a continuous glutamine polypeptide as defined in claim 1, wherein: the low-temperature preservation in the step 4) is 10 ℃.
5. The method for producing a continuous glutamine polypeptide as defined in claim 1, wherein: the diameter of the flash medium-pressure column in the step 5) is 20-30mm, and a column temperature box is added at 10 ℃.
6. The method for producing a continuous glutamine polypeptide as defined in claim 1, wherein: the leaching speed of the step 5) is 17-22ml/min, and the pH value is measured until the pH value is acidic.
7. A method of producing a continuous glutamine polypeptide as defined in claim 1, characterized by: the reversed phase silica gel column in the step 6) has the particle size of 10um and the aperture of 300A, and the column temperature is increased to 20 ℃.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002064619A2 (en) * | 2001-02-09 | 2002-08-22 | Massachusetts Institute Of Technology | Peptides that inhibit poly-glutamine aggregation |
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| US7067321B2 (en) * | 2002-02-22 | 2006-06-27 | University Of Tennessee Research Foundation | Polyglutamine repeat sequences |
| US10570177B2 (en) * | 2017-03-01 | 2020-02-25 | The Chinese University Of Hong Kong | P3 peptidylic inhibitors for treating CAG-repeat RNA toxicity in polyglutamine diseases |
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| WO2010115033A2 (en) * | 2009-04-02 | 2010-10-07 | Regents Of The University Of Minnesota | Nucleotide repeat expansion-associated polypeptides and uses thereof |
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