Method for large-scale purification of fusion proteins containing histaq and formate hydrolysis sites

A fusion protein, large-scale technology, applied in the field of protein purification, can solve the problems of low yield and increased by-products, and achieve the effects of high yield, short time consumption and high purity

Inactive Publication Date: 2014-10-08
广州暨南大学科技园管理有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is currently no report about the use of formic acid hydrolysis to hydrolyze fusion proteins on a large scale to prepare polypeptides. This may be due to the poor control of formic acid hydrolysis conditions and easy to cause non-specific shearing (Expression, purification and structural studies of ahort antimicrobial peptide. Biochimica et Biophsica Acta: 1788 (2009) 314-323), resulting in increased by-products, resulting in low yield

Method used

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  • Method for large-scale purification of fusion proteins containing histaq and formate hydrolysis sites
  • Method for large-scale purification of fusion proteins containing histaq and formate hydrolysis sites
  • Method for large-scale purification of fusion proteins containing histaq and formate hydrolysis sites

Examples

Experimental program
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Effect test

Embodiment 1

[0058] (1) Pretreat the fermentation broth of the engineered bacteria expressing the fusion protein containing His Taq and formic acid hydrolysis sites to obtain a crude extract of the fusion protein containing His Taq and formic acid hydrolysis sites:

[0059] 2100ml of PBS buffer with pH 7.4 and 50mM was added to 210g of wet bacterial cells (obtained from fermentation in Example 1 of Patent Application No. 201010221511.8) and mixed uniformly to obtain a bacterial cell suspension. The bacterial suspension is broken once in a high-pressure homogenizer (pressure 150MPa). The supernatant was discarded by centrifugation at 4°C at 10,000 rpm for 30 minutes, and the pellet was taken. The sample was present in the pellet as inclusion bodies. Wash the precipitate with 2100ml washing solution A (pH 8.0, 20mM Tris-HCl), and the washing method is 50rpm / min stirring for 10min; then centrifuge at 4°C 10000rpm for 30min to discard the supernatant and take the precipitate. Wash the pellet wit...

Embodiment 2

[0088] (1) Pretreating the fermentation broth of the engineered bacteria expressing the fusion protein containing His Taq and formic acid hydrolysis sites to obtain a crude extract of the fusion protein containing His Taq and formic acid hydrolysis sites: Same as step (1) in Example 1, The only difference is that the sample is 300g of bacteria obtained from fermentation in Example 2 of Patent Application No. 201010221511.8, and the volume of the solution used is 10 times the mass of the bacteria. The washed precipitate is washed with 20mM imidazole-containing dissolution buffer A (6M Urea, 50mM Na 2 HPO 4 , 500mM NaCl, pH 8.0) dissolved.

[0089] (2) Initial purification of crude fusion protein extract

[0090] A. Equilibrate the Ni-NTA column with 20mM imidazole-containing lysis buffer A (same step (1)) to stabilize the baseline, and then load the crude fusion protein extract obtained in step (1) onto the column at a flow rate of 4ml / min, Ni -The loading capacity of the NTA colum...

Embodiment 3

[0107] (1) Pretreating the fermentation broth of the engineered bacteria expressing the fusion protein containing His Taq and formic acid hydrolysis sites to obtain a crude extract of the fusion protein containing His Taq and formic acid hydrolysis sites: Same as step (1) in Example 1, The only difference is that the sample is 370 g of the bacteria obtained by fermentation in Example 3 of Patent Application No. 201010221511.8, the pressure of the high-pressure homogenizer is 120 MPa, and the volume of the solution used is 10 times the mass of the bacteria.

[0108] (2) Initial purification of crude fusion protein extract

[0109] A. Equilibrate the Ni-NTA column with 10mM imidazole-containing lysis buffer A (same step (1)) until the baseline is stable, and then load the crude fusion protein extract obtained in step (1) on the column at a flow rate of 6ml / min. -The loading capacity of the NTA column is 24.9mg protein / ml column volume; after loading the column, wash the column to bas...

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Abstract

The invention discloses a method for purifying a fusion protein containing a histidine tag (His Tag) and a formic acid hydrolysis site on a large scale and application thereof. The method comprises the following steps of: pretreating fermentation liquor of engineering bacteria for expressing the fusion protein containing the His Tag and the formic acid hydrolysis site to obtain fusion protein crude extract containing the His Tag and the formic acid hydrolysis site; performing affinity chromatography by using a nickel-nitrilotriacetic acid (Ni-NTA) column and desalting by using a C18 reverse hydrophobic column to obtain the primarily purified protein of the fusion protein crude extract; hydrolyzing formic acid, and performing affinity chromatography by using the Ni-NTA column and desalting by using the C18 reverse hydrophobic column to obtain a primarily purified target polypeptide; and refining by high performance liquid chromatography (HPLC) to obtain a target polypeptide with the purity of over 95 percent. By the method, the fusion protein containing the His Tag and the formic acid hydrolysis site is effectively prepared on a large scale, the target polypeptide has high yield and purity and low cost, time consumption is low, the production scale of above g level can be achieved, and the requirements of animal experiments and preclinical experimental research can be met.

Description

Technical field [0001] The invention relates to a protein purification method, in particular to a method and application for large-scale purification of fusion proteins containing His Taq and formic acid hydrolysis sites. Background technique [0002] At present, the preparation methods of polypeptides mainly include chemical synthesis and genetic engineering. For polypeptides with a small number of amino acids and small requirements, they can be synthesized by chemical synthesis. However, it is difficult to chemically synthesize peptides with a large number of amino acids, and the cost is high, and the cycle is also long. For example, 100mg samples with a purity greater than 95% require about 420 yuan per amino acid, and about 50 amino acids require more than 20,000 yuan. , And the purity is greater than 98% requires 800 yuan for each amino acid, and 40,000 yuan for 50 amino acids, and it takes about 4 weeks. Therefore, in order to obtain a large amount of high purity and a la...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/22C07K1/20C07K1/12
Inventor 李弘剑郑飞冉艳红苏正定周天鸿
Owner 广州暨南大学科技园管理有限公司
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