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Method for purifying lysostaphin by antibody affinity chromatography

A lysostaphin enzyme and a chromatographic purification technology are applied in the field of chromatographic purification of lysostaphinase, and can solve the problems of short column efficiency of affinity adsorption column, low adsorption capacity, and inability to be applied to industrialized production and the like

Active Publication Date: 2008-11-12
昆山博青生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the affinity adsorption column made with bacterial cells or cell wall peptidoglycan as a ligand has a very short column efficiency, basically loses the affinity effect after being used for 2-3 times, and the adsorption capacity is low, so it cannot be applied to industrial production

Method used

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  • Method for purifying lysostaphin by antibody affinity chromatography
  • Method for purifying lysostaphin by antibody affinity chromatography
  • Method for purifying lysostaphin by antibody affinity chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Purification of monoclonal antibody to lysostaphin:

[0049] Chromatography medium: 5ml protein A hightrap prepacked column, purchased from Pharmacia Biotechnology Co., Ltd.

[0050] Monoclonal antibody sample: Using lysostaphin as an antigen, immunize BalB / c mice according to the conventional monoclonal antibody preparation method to prepare 15ml of mouse ascites containing lysostaphin monoclonal antibody. Wherein, the content of lysostaphin monoclonal antibody is 0.16mg / ml ;

[0051] Chromatography equipment: AKTA explorer 100 protein purification development system, purchased from Pharmacia Biotechnology Co., Ltd.

[0052] Equilibration and washing buffer: PBS buffer at pH 8.0.

[0053] Elution buffer: Gly-HCl buffer at pH 2.8 containing 0.1 mol / L glycine.

[0054] Equilibrium process: balance 5ml protein A prepacked column with PBS buffer solution with pH 8.0, pass it into the prepacked column at a flow rate of 3ml per minute, the amount is 6 times the volume o...

Embodiment 2

[0061] Purification of Lysostaphin Polyclonal Antibody:

[0062] Chromatography medium: 5ml protein A hightrap prepacked column, purchased from Pharmacia Biotechnology Co., Ltd.

[0063] Polyclonal antibody sample: Using lysostaphin as an antigen, New Zealand rabbits were immunized according to the conventional polyclonal antibody preparation method, and 8ml of rabbit antiserum containing lysostaphin polyclonal antibody was prepared. Wherein, the content of lysostaphin polyclonal antibody is 2.75mg / ml :

[0064] Chromatography equipment: AKTA explorer 100 protein purification development system, purchased from Pharmacia Biotechnology Co., Ltd.

[0065] Equilibrium and washing buffer: Tris-HCl buffer at pH 8.0 containing 0.1mol / LTris.

[0066] Elution buffer: Gly-HCl buffer at pH 2.8 containing 0.1 mol / L glycine.

[0067] Equilibrium process: equilibrate 5ml protein A prepacked column with Tris-HCl buffer solution containing 0.1mol / L Tris at pH 8.0, the flow rate is 4ml pe...

Embodiment 3

[0073] Purification of Lysostaphin by Affinity Chromatography

[0074] Affinity medium: a chromatographic column filled with 3 ml of Sepharose 4B gel (purchased from Pharmacia Biotechnology Co., Ltd., activated by CNBr) coupled with the monoclonal antibody prepared in Example 1.

[0075] Sample: 10ml of Escherichia coli culture supernatant containing lysostaphin gene prepared according to Chinese patent CN1900290, containing 964 units of lysostaphin, adding an equal volume of 0.1mol / L Tris-HCl at pH 8.0 containing 0.15mol / L NaCl Buffer, prepare 20ml of sample solution.

[0076] Chromatography equipment: AKTA explorer 100 protein purification development system, purchased from Pharmacia Biotechnology Co., Ltd.

[0077] Equilibrium and washing buffer: Tris-HCl buffer with pH 8.0 containing 0.1mol / L NaCl;

[0078] Elution buffer: pH 4.3 acetate buffer containing 0.1 mol / L sodium acetate.

[0079] Neutralization buffer: Tris-HCl buffer at pH 8.0 containing 1mol / LTris.

[0080]...

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Abstract

The invention discloses a method for purifying lysostaphin by antibody affinity chromatography, comprising the following steps of: (1) cleaning and eluting an antibody sample containing lysostaphin by passing the antibody sample through a chromatographic column which is filled with protein A resin and is balanced by buffer liquid A; collecting and drying by freezing eluting peak; (2) mixing the freeze-dried product with an affinity chromatography medium to form an immunized affinity chromatography medium; (3) making the lysostaphin sample pass through the chromatography column which is filled with immunoaffinity chromatography medium and is previously balanced with the buffer liquid A; then cleaning the sampled immunoaffinity chromatography column with the buffer liquid A; and eluting the lysostaphin from the chromatography column with a buffer liquid C; and collecting the eluting peak. The method has the advantages of rapid processing speed, high recycling rate and good repeatability, long column efficiency and basically invariable joint efficiency, along with a recycling rate of an active protein of between 85 and 95 percent, a specific activity of more than 1000 U / mg, a multiple of protein purification of more than between 3 and 5 times, and a purity of between 95 and 98 percent.

Description

technical field [0001] The invention relates to an enzyme purification method, in particular to a chromatographic purification method for lysostaphin. Background technique [0002] Lysostaphin is an extracellular protein product secreted by Staphylococus Simulans, with a molecular weight of 27kDa and 246 amino acids. It is an endopeptidase. The enzyme can cut the five-glycine peptide bond bridge structure in the peptidoglycan of staphylococcal cell wall, so as to achieve the purpose of rapidly dissolving and killing bacteria. Even for drug-resistant Staphylococcus aureus, MRSA and "super bacteria", lysostaphin also has a strong bactericidal effect, and it is not easy to induce drug-resistant strains, so it is expected to become a new type of organism for the treatment of drug-resistant bacterial infections preparation. [0003] In 1987, Fudan University successfully expressed lysostaphin in Bacillus sphaericus, and established a separation and purification method for the e...

Claims

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Application Information

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IPC IPC(8): C12N9/36
Inventor 黄晋江张继恩黄青山
Owner 昆山博青生物科技有限公司
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