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α-Hydroxy Acid-Based Protein Purification Methods

A protein purification and hydroxy acid technology, which is applied in the direction of peptide preparation methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as inapplicability, improve purification efficiency and yield, overcome high costs, reduce The effect of time and economic cost

Active Publication Date: 2018-11-02
COFCO NUTRITION & HEALTH RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in such methods, there is still a lack of means to effectively control the self-assembly process
In addition, most of the existing methods remove short peptides by protease digestion or chemical cleavage, so there are certain requirements for the sequence characteristics of the target protein
[0005] Although it is known in the field of chemical engineering that protecting groups can be used to protect the protein sequence during the total synthesis of proteins and remove the protecting groups after the synthesis is completed, however, since almost all types of organisms on the earth use 20 natural amino acids for in vivo The biosynthesis of protein, the protein synthesis using the cell's own synthesis system does not apply the above principles

Method used

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  • α-Hydroxy Acid-Based Protein Purification Methods
  • α-Hydroxy Acid-Based Protein Purification Methods
  • α-Hydroxy Acid-Based Protein Purification Methods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1 Expression and purification of fusion protein using wild-type defensin 5 (Defensin 5) as the target protein

[0119] The solutions and medium components used in Example 1 are as follows.

[0120] 1x PBS buffer:

[0121] NaCl: 7.9g (Beijing Chemical Plant, purity ≥99.5%, pH: 5.0-8.0); KCl: 0.2g (Beijing Chemical Plant, purity ≥99.5%, pH: 5.0-8.0);

[0122] K H 2 PO 4 : 0.24g (Beijing Chemical Plant, purity ≥ 99.5%, pH: 4.2 ~ 4.5);

[0123] K 2 HPO 4 : 1.8g (Beijing Chemical Plant, purity ≥ 99.0%, pH: 8.9 ~ 9.4);

[0124] Dissolve in 800ml of distilled water, and finally add distilled water to make up to 1L, and adjust the pH value of the solution to 7.4 with HCl (analytical grade, Sinopharm Chemical Reagent Co., Ltd.).

[0125] Luria-Bertani (LB) liquid medium:

[0126] Peptone (Fisher Scientific) 10g / L;

[0127] NaCl (Fisher Scientific) 10g / L;

[0128] Yeast powder (Fisher Scientific) 5g / L;

[0129] pH=7.

[0130] Autoclave at 121°C for 20 minutes....

Embodiment 2

[0166] Example 2 Expression and purification of fusion protein using wild-type histidine-rich peptide (Histatin) as target protein

[0167] Unless otherwise specified, the buffer solution, culture medium, enzyme, initial plasmid and assay method used in Example 2 are all the same as in Example 1.

[0168] LB liquid and solid media were used in plasmid construction, strain cultivation, maintenance, and induction.

[0169] 1. Construction of expression strains

[0170] In this example, the wild-type histidine-rich peptide (Histatin) (GenBank: NM_002159.3) was used as the target protein. Hisstatin has antibacterial activity. The activity of the protein can be visually detected by dropping it on the surface of a plate coated with bacteria and observing the size of the inhibition zone.

[0171] In this example, ABZ (SEQ ID NO: 2) with self-assembly function is used as a functional fragment. Since ABZ has self-assembly function, it can aggregate in the expression cell to form insol...

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Abstract

The present invention relates to a method for purifying a target protein based on alpha-hydroxy acid. According to the method, specifically alpha-hydroxy acid and a functional fragment are linked to a target protein, purification is performed with the functional fragment, and the alpha-hydroxy acid is hydrolyzed by changing the pH value so as to obtain the target protein. According to the present invention, the purification method is suitable for the purification of the proteins expressed by a variety of expression systems, has advantages of low cost, simple process, easy industrial achievement, can be used for the high-throughput protein purification on the laboratory scale, and can further be used for the low-cost protein production on the industrial-scale.

Description

technical field [0001] The invention relates to a method for purifying target protein based on α-hydroxy acid. Specifically, the present invention obtains the target protein by linking the α-hydroxy acid and the functional fragment to the target protein, purifying the functional fragment, and then changing the pH conditions to cause cleavage (hydrolysis) at the α-hydroxy acid. Background technique [0002] Whether it is in the laboratory scale or in the field of industrial production, protein purification is a key link in genetic engineering and protein engineering. According to statistics, the cost of separation and purification of genetic engineering products accounts for about 60% to 80% of the total cost (Chen Hao, Chen Yuhong, Zhu Dexu, Liu Jianning, Recombinant Protein Purification Technology, China Biotechnology Journal, 2002, 22 (5): 87-92). Efficient prokaryotic, fungal and eukaryotic protein expression systems have been developed. However, no matter what kind of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/00C07K19/00C07K1/14C12N1/21C12N1/15C12N1/19C12N5/10
CPCC07K14/4703C07K14/4723C07K2319/20C12N9/93C12Y601/01026
Inventor 安泰王磊陈博杨鑫朱威宇朱镜羲林海龙牛兴和郝小明
Owner COFCO NUTRITION & HEALTH RES INST
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