Method for purifying and preparing antibacterial peptide

Abstract

The invention discloses a method and device for purifying and preparing an antibacterial peptide. The method and device solve the problem that the prior art cannot guarantee a yield of 50% or more while the purity is greater than 99% in antibacterial peptide purification. The method comprises: 1, preparing a crude antibacterial peptide solution, 2, adding the crude antibacterial peptide solution into a semipermeable membrane, and orderly carrying out dialysis through a buffer and purified water to obtain an antibacterial peptide primary pure solution, 3, refining the antibacterial peptide primary pure solution through reverse phase chromatography to obtain an antibacterial peptide refined pure solution, and 4, transferring salts in the antibacterial peptide refined pure solution through anHPLC method to obtain an antibacterial peptide solution and carrying out reduced pressure concentration and drying to obtain an antibacterial peptide finished product. The method can promote the semi-permeable membrane purification and the reverse chromatography purification and realizes a yield of 50% or more while the purity is greater than 99%.

Description

technical field

[0001] The invention relates to the field of purification, in particular to a method for purifying and preparing antimicrobial peptides. Background technique

[0002] Antimicrobial peptides are a class of polypeptides with antibacterial activity. By acting on the bacterial cell membrane, destroying its integrity and producing perforation, it enters the cell and destroys its organelles to cause metabolic disorders. Bacterial contents leak out of the cell causing bacterial death. Antimicrobial peptides not only have a good bactericidal effect on bacteria and fungi, but also have antiviral activity and promote wound healing. At the same time, the combination of antimicrobial peptide and lysozyme also has a synergistic antibacterial effect. Under the action of these two core components, a double wall-breaking and bactericidal effect on pathogenic bacteria is formed.

[0003] The antimicrobial peptide is similar to MSI-78 in foreign clinical trials, which has c...

Examples

Embodiment 1

[0037] A method for purifying and preparing antimicrobial peptides, comprising:

[0038] (1) Sample treatment: The antimicrobial peptide crude peptide with a chromatographic purity of more than 50% was obtained by solid-phase synthesis, dissolved in purified water at a concentration of 50g / L, stirred, and filtered with a 0.45um water filter membrane after complete dissolution. Remove the insoluble matter to obtain the filtrate, which is the crude antimicrobial peptide after drying.

[0039] (2) Initial purification: Add 3.2 L of purified water to 160 g of antimicrobial peptide crude product to prepare a 50 mg / ml antimicrobial peptide crude product solution, and then filter with a microporous membrane to obtain a crude antimicrobial peptide solution for later use.

[0040] Put the antimicrobial peptide crude product solution into an RC semipermeable membrane with a molecular weight cut-off of 500 Daltons, about 60% of the total capacity volume, into 32L acetate buffer solution,...

Embodiment 2

[0048] This example is a comparative example of Example 1. In this example, the purification method and conditions in the comparative document CN201511014180.X are used to treat the antimicrobial peptide. The treatment process and results are as follows:

[0049] Step 1. Take 5 g of antibacterial peptide crude peptide, add 50 ml of 1% acetic acid solution, and prepare 10% antibacterial peptide crude peptide solution.

[0050] Step 2, the acetic acid solution of antimicrobial peptide is loaded into is that 500 Dalton RC semi-permeable membranes are packed in molecular weight cut-off, and loading is 70% of semi-permeable membrane loading volume, is placed in 2L acetate buffer (pH3.5 ) buffer solution, stirred and diafiltered at 20-30°C until the buffer solution pH=2.0-3.0.

[0051] Step 3. Transfer the semipermeable membrane of the acetic acid solution containing the antimicrobial peptide to 3L of purified water, stir and diafilter at 20-30° C. until the pH of the solution in the ...

Embodiment 3

[0064] The present embodiment is the comparative example of embodiment 1, only adopts the reversed-phase chromatography in embodiment 1 to purify in the present embodiment, concrete process and experimental result are as follows:

[0065] (1) Sample treatment: Dissolve the antimicrobial peptide in purified water at a concentration of 50mg / mL, stir, and filter it with a 0.45um water filter membrane after completely dissolving to remove insoluble matter to obtain the filtrate, which is the crude antimicrobial peptide after drying .

[0066] (2) Initial purification: add purified water to 2 g of antimicrobial peptide crude product to prepare a 35% antimicrobial peptide crude product solution, then filter with a microporous membrane to obtain the antimicrobial peptide crude product solution for later use.

[0067] (3) pure

[0068] Reversed-phase chromatographic conditions: a high-performance liquid chromatography preparative column with octadecylsilane bonded silica gel as the s...

technical field

[0001] The invention relates to the field of purification, in particular to a method for purifying and preparing antimicrobial peptides. Background technique

[0002] Antimicrobial peptides are a class of polypeptides with antibacterial activity. By acting on the bacterial cell membrane, destroying its integrity and producing perforation, it enters the cell and destroys its organelles to cause metabolic disorders. Bacterial contents leak out of the cell causing bacterial death. Antimicrobial peptides not only have a good bactericidal effect on bacteria and fungi, but also have antiviral activity and promote wound healing. At the same time, the combination of antimicrobial peptide and lysozyme also has a synergistic antibacterial effect. Under the action of these two core components, a double wall-breaking and bactericidal effect on pathogenic bacteria is formed.

[0003] The antimicrobial peptide is similar to MSI-78 in foreign clinical trials, which has c...

Examples

Embodiment 1

[0037] A method for purifying and preparing antimicrobial peptides, comprising:

[0038] (1) Sample treatment: The antimicrobial peptide crude peptide with a chromatographic purity of more than 50% was obtained by solid-phase synthesis, dissolved in purified water at a concentration of 50g / L, stirred, and filtered with a 0.45um water filter membrane after complete dissolution. Remove the insoluble matter to obtain the filtrate, which is the crude antimicrobial peptide after drying.

[0039] (2) Initial purification: Add 3.2 L of purified water to 160 g of antimicrobial peptide crude product to prepare a 50 mg / ml antimicrobial peptide crude product solution, and then filter with a microporous membrane to obtain a crude antimicrobial peptide solution for later use.

[0040] Put the antimicrobial peptide crude product solution into an RC semipermeable membrane with a molecular weight cut-off of 500 Daltons, about 60% of the total capacity volume, into 32L acetate buffer solution,...

Embodiment 2

[0048] This example is a comparative example of Example 1. In this example, the purification method and conditions in the comparative document CN201511014180.X are used to treat the antimicrobial peptide. The treatment process and results are as follows:

[0049] Step 1. Take 5 g of antibacterial peptide crude peptide, add 50 ml of 1% acetic acid solution, and prepare 10% antibacterial peptide crude peptide solution.

[0050] Step 2, the acetic acid solution of antimicrobial peptide is loaded into is that 500 Dalton RC semi-permeable membranes are packed in molecular weight cut-off, and loading is 70% of semi-permeable membrane loading volume, is placed in 2L acetate buffer (pH3.5 ) buffer solution, stirred and diafiltered at 20-30°C until the buffer solution pH=2.0-3.0.

[0051] Step 3. Transfer the semipermeable membrane of the acetic acid solution containing the antimicrobial peptide to 3L of purified water, stir and diafilter at 20-30° C. until the pH of the solution in the ...

Embodiment 3

[0064] The present embodiment is the comparative example of embodiment 1, only adopts the reversed-phase chromatography in embodiment 1 to purify in the present embodiment, concrete process and experimental result are as follows:

[0065] (1) Sample treatment: Dissolve the antimicrobial peptide in purified water at a concentration of 50mg / mL, stir, and filter it with a 0.45um water filter membrane after completely dissolving to remove insoluble matter to obtain the filtrate, which is the crude antimicrobial peptide after drying .

[0066] (2) Initial purification: add purified water to 2 g of antimicrobial peptide crude product to prepare a 35% antimicrobial peptide crude product solution, then filter with a microporous membrane to obtain the antimicrobial peptide crude product solution for later use.

[0067] (3) pure

[0068] Reversed-phase chromatographic conditions: a high-performance liquid chromatography preparative column with octadecylsilane bonded silica gel as the s...

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