Method for preparing high-purity apolipoprotein A-I

An apolipoprotein and A-I technology, which is applied to the preparation field of apolipoprotein A-I, can solve the problems of unsuitable industrial production, low protein yield, flammability and explosion, etc., and is suitable for large-scale industrial production and has good protein activity. , the effect of high yield

Active Publication Date: 2008-06-25
SHANGHAI RAAS BLOOD PRODUCTS CO LTD
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AI Technical Summary

Problems solved by technology

[0003] At present, the commonly used preparation methods for obtaining apoA-I include ultracentrifugation, organic solvent precipitation, and high-performance liquid phase. Although high-purity apoA-I can be obtained, it has some obvious disadvantages: ①The preparation amount is small, generally only Milligram-level apoA-I protein can be obtained, which is not suitable for industrial production; ②The protein yield is low, and most of the apoA-I is lost in the preparation process after multi-step treatment such as ultracentrifugation, organic solvent precipitation, and column chromatography; ③ High cost, requiring expensive instruments such as ultracentrifuges; ④ poor safety, organic solvents such as ethanol, acetone, and trichloroacetic acid are not only physiologically toxic, but also flammable and explosive, which is not conducive to safe production

Method used

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  • Method for preparing high-purity apolipoprotein A-I
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  • Method for preparing high-purity apolipoprotein A-I

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preparation example Construction

[0049] For the method for preparing apoA-I provided by the invention, refer to the attached figure 1 A flow chart, which includes the following steps:

[0050] (1) Component four precipitation pretreatment

[0051]The fourth component is the human plasma obtained by the cohn low-temperature ethanol method (Cohn E J, StrongLE, Huges WL, et al. Preparation and properties of serum and plasma proteins IVA system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. Amer Chem Soc, 1946, 68: 459-475.). Dissolve the precipitate of component four in the buffer solution, then add a certain concentration of urea, and mix thoroughly to denature the proteins such as apoA-I in component four. The so-called denaturation is reversible.

[0052] The concentration of the added urea is 1-8M, preferably 3-7M, more preferably 5-6M. The weight ratio of component four precipitation to the added urea is 1:30-300, preferably 1:90-240, more pr...

Embodiment 1

[0085] Preparation of apoA-I①

[0086] 0.2 g of component four, the chromatographic column adopts 5ml DEAE anion exchange prepacked column (purchased from GE Healthcare company), the purification equipment is AKTA EXPLORER 100 (purchased from GE Healthcare company), and the conductometric measurement adopts the conductivity detection device that AKTA EXPLORER 100 comes with .

[0087] (1) Component four precipitation pretreatment

[0088] 0.2 g of component four precipitate was dissolved in 100 ml of Tris buffer solution with pH 7.8 at 4°C, and urea was added to a final concentration of 6 mol / L, and mixed well. Centrifuge at 8000 rpm to remove insoluble impurities, and then filter with a 0.45 μm membrane filter.

[0089] (2) The first DEAE anion exchange column chromatography

[0090] The DEAE column is balanced with Tris buffer solution (pH7.8) containing 6mol / L urea, and the filtrate of step (1) flows through the DEAE anion exchange chromatography column at a flow rate of...

Embodiment 2

[0098] Preparation of apoA-I②

[0099] Take by weighing 0.2 grams of component four, and the experimental equipment is the same as in Example 1.

[0100] (1) Component four precipitation pretreatment

[0101] 0.2 g of the precipitate of component four was dissolved in 100 ml of Tris buffer at pH 7.8 at 4°C, and urea was added to a final concentration of 1 mol / L, and thoroughly mixed. Centrifuge at 8000 rpm to remove insoluble impurities, and then filter with a 0.45 μm membrane filter.

[0102] (2) The first DEAE anion exchange column chromatography

[0103] The DEAE column was balanced with Tris buffer (pH7.8) containing 1mol / L urea, and the filtrate from step (1) flowed through the DEAE anion-exchange chromatography column at a flow rate of 1ml / min, and apoA-I and miscellaneous proteins were bound to the column.

[0104] Elute apoA-I with Tris buffer containing 1mol / L urea (conductivity 3.5ms / cm, pH7.8), the eluate volume is about 20ml; then wash with Tris buffer pH 7.8 co...

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Abstract

The invention discloses a method for obtaining the apolipoprotein A-I, which comprises the steps that: a. Component 4 obtained through the Cohn cold ethanol method is mixed with the 1-8M urea so as to form the Component 4 pretreatment solution; b. the Component 4 pretreatment solution obtained in Step a flows through an anionic exchange chromatography column before the chromatography column is washed using the buffer containing the 1-8M urea so as to obtain the preliminarily purified apolipoprotein A-I; c. the solution containing the preliminarily purified apolipoprotein A-I obtained in Step b flows through the anionic exchange chromatography column and the chromatography column is washed using the buffer containing the 0-1M urea after the impurities are eluted so as to obtain the purified apolipoprotein A-I. The method delives a high protein extraction yield, has a low cost and is suitable for industrialized production, and simultaneously the method also sufficiently utilzies the plasma resources.

Description

technical field [0001] The present invention relates to protein preparation, in particular to a preparation method of apolipoprotein A-I. Background technique [0002] Apolipoprotein A-I (Apolipoprotein A-I, apoA-I) is the main apolipoprotein of high-density lipoprotein (HighDensity Lipoprotein, HDL), which is a single polypeptide chain consisting of 243 amino acid residues and a molecular weight of 28.3kD. The main function of HDL is to participate in reverse cholesterol transport (Reverse Cholesterol Transport, RCT), moving cholesterol in peripheral tissue cells and transporting it to the liver for conversion and elimination, so it plays an important role in combating the occurrence and development of atherosclerosis (AS). apoA-I is the main bearer of the anti-AS function of HDL. apoA-I also has anti-inflammatory and anti-endotoxin functions, so it is one of the focuses of lipid metabolism research. Since apoA-I has the characteristics of liver targeting, it also has a g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/18C07K14/775
CPCC07K14/775A61K38/16C07K1/18
Inventor 黄凯何秋许必雄包骧飞李军辉沈积慧李春洲
Owner SHANGHAI RAAS BLOOD PRODUCTS CO LTD
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