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Purifying method for neutral hydrophobic polypeptide

A purification method and hydrophobic technology, applied in the field of peptide purification, can solve problems such as difficulty, limited selectivity of chromatographic columns, poor separation effect of normal phase chromatography, etc., achieve good pretreatment effect, reduce production cost, and solve extremely low yield Effect

Active Publication Date: 2018-12-21
GL BIOCHEM SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for hydrophobic peptides, especially neutral hydrophobic peptides that mostly contain Ala, Ile, and Val raw materials, there are still problems that cannot be purified because there is no way to completely dissolve the peptide, or because of the selected dissolution method or mobile phase. The yield of neutral hydrophobic peptides is extremely low, and even the high purity requirements cannot be met.
Nowadays, most neutral and hydrophobic peptides are dissolved in DMSO or strong acid and strong base. However, if the viscosity of DMSO is too high, it is basically difficult for the peptide to be adsorbed on the chromatographic column. Peptides dissolved in strong acid and strong base will not only reduce the service life of the chromatographic column , and most of the peptides will be taken out of the column by strong acid and strong base, resulting in a very small amount of elution separation
In the prior art, normal phase chromatography is used to separate hydrophobic polypeptides, but the separation effect of normal phase chromatography is very poor, and the selectivity of the chromatographic column is also limited. It is very difficult to obtain high-purity neutral hydrophobic polypeptides.
[0004] The present invention proposes to solve the existing defects and specifically solve the current problem of difficult purification of neutral hydrophobic polypeptides

Method used

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  • Purifying method for neutral hydrophobic polypeptide
  • Purifying method for neutral hydrophobic polypeptide
  • Purifying method for neutral hydrophobic polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1. Taking the weak neutral hydrophobic polypeptide sequence KDLL KDLL KDLL KDLL as an example, the specific implementation steps are as follows:

[0046] 1) Weigh 300mg of polypeptide sample, add 30ml of acetone to dilute, oscillate and stir to clarify, pass through a PE column with 40g of diatomaceous earth and 10g of silica gel filler, filter to obtain the clear liquid, and set aside.

[0047] 2) Proportioning acetone and perchloric acid aqueous solution as the mobile phase, wherein phase A is pure acetone solution, and phase B is aqueous perchloric acid solution containing 0.15% by mass.

[0048] 3) The chromatographic column is a chromatographic column C18 with ordinary silica gel-bonded stationary phase packing, with a diameter of 30mm, a length of 250mm, and a pore size of 300A; the flow rate of the high-pressure pump is set to 30ml / min, and the wavelength of the detector is 220nm.

[0049] 4) Rinse the chromatographic column and instrument with pure methanol for ...

Embodiment 2

[0057] 1. Taking the weak neutral hydrophobic polypeptide sequence IIREEIIRIIRECQIIC as an example, the specific implementation steps are as follows:

[0058] 1) Weigh 500mg of polypeptide sample, add 40ml of isobutanol to dilute, shake and stir, pass through a PE column filled with 30g of diatomaceous earth and 20g of silica gel, filter to obtain the clear liquid, and set aside.

[0059] 2) Proportioning isobutanol and ammonium sulfate aqueous solution as the mobile phase, phase A is pure isobutanol solution, and phase B is ammonium sulfate aqueous solution containing 0.18% by mass.

[0060] 3) The chromatographic column is a chromatographic column C8 (or C4) with ordinary silica gel-bonded stationary phase packing, with a diameter of 40mm, a length of 300mm, and a pore size of 300A; the flow rate of the high-pressure pump is set at 40ml / min, and the wavelength of the detector is 220nm.

[0061] 4) Rinse the chromatographic column and instrument with pure methanol for 30-50 m...

Embodiment 3

[0070] 1. Taking the strongly neutral hydrophobic polypeptide sequence VFVVFVVFVVVFVVHVVD as an example, the specific implementation steps are as follows:

[0071] 1) Weigh 500mg of polypeptide sample, add 10ml of hexafluoroisopropanol to fully dissolve the polypeptide, pass through a PE column filled with 20g of diatomaceous earth and 15g of silica gel, filter to obtain the clear liquid, and set aside.

[0072] 2) Proportioning trifluoroethanol and aqueous hydrochloric acid as the mobile phase, phase A is pure trifluoroethanol solution, and phase B is aqueous hydrochloric acid containing 0.05% by mass. Linear gradient 10-60%.

[0073] 3) A chromatographic column (C18) based on polystyrene divinylbenzene as a stationary phase filler, with a diameter of 30mm, a length of 250mm, and a pore size of 120A. The flow rate of the high-pressure pump is set at 35ml / min, and the wavelength of the detector is 220nm.

[0074] 4) Rinse the chromatographic column and instrument with pure m...

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Abstract

The invention discloses a purifying method for neutral hydrophobic polypeptide, and the purifying method is mainly used for solving the problem that neutral hydrophobic polypeptide is difficult to purify, a high-purity product is difficult to obtain, and the product yield is extremely low. The purifying method comprises the steps of sorting neutral hydrophobic polypeptide according to strength, selecting a dissolving method, selecting a chromatographic column, selecting an analysis and preparation flow phase, and eluting and collecting. A sequence of the neutral hydrophobic polypeptide comprises polypeptide of Ala, Ile, Val and Leu raw material; the dissolving method for the neutral hydrophobic polypeptide can select one of hexafluoroisopropanol or urea to dissolve, and also can select oneof acetone or isobutanol to greatly dilute, oscillate and stir, and a PE column manufactured from diatomite and a silica filler in different proportion is used for filtering to obtain clear liquor; the chromatographic column can be a chromatographic column of a fixed-phase filler bonded by silica gel or a chromatographic column of the fixed-phase filler which takes polystyrene divinylbenzene as matrix; the analysis and preparation flow phase select a special system according to different dissolving methods; and eluting and collecting for hydrophobic polypeptide are performed.

Description

technical field [0001] The invention relates to the technical field of polypeptide purification, in particular to a purification method for neutral hydrophobic polypeptides. Background technique [0002] Biologically active polypeptide is a general term for different peptides composed of 20 natural amino acids in different compositions and arrangements, ranging from dipeptides to complex linear and circular structures. It is a multifunctional compound derived from proteins. Many active substances in the human body exist in the form of peptides. Polypeptides are involved in the fields of hormones, nerves, cell growth and reproduction of the human body. Its importance lies in regulating the physiological functions of various systems and cells in the body, activating relevant enzymes in the body, promoting the permeability of intermediate metabolic membranes, or controlling DNA transcription or Affect specific protein synthesis, and finally produce specific physiological effec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/36C07K1/20C07K1/34
CPCC07K1/20C07K1/34C07K1/36
Inventor 徐红岩闫凤
Owner GL BIOCHEM SHANGHAI
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