Method for purifying regrouped chicken colibacillosis outer membrane protein inclusion body and carrying out renaturation by utilizing high-efficiency renaturation liquid

A technology of chicken Escherichia coli and outer membrane protein, which is applied in the downstream protein purification field of biomedicine and bioengineering, can solve the problems of long cycle, complicated operation steps, low recovery rate, etc., and achieve improved renaturation rate, simple equipment requirements, and improved The effect of renaturation rate

Inactive Publication Date: 2015-05-20
TIANJIN SHENGJI GRP CO LTD
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Problems solved by technology

[0006] In order to overcome the shortcomings of the existing inclusion body renaturation method in the production of outer membrane protein inclusion body, which requires complex operation steps, long cycle and low recovery rate, a purification and high-efficiency method for recombinant chicken Escherichia coli outer membrane protein inclusion body is provided. The refolding method of refolding liquid

Method used

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Embodiment Construction

[0015] In order to achieve the above purpose, a method for purifying recombinant chicken Escherichia coli outer membrane protein inclusion body and high-efficiency refolding solution for refolding it was designed. The specific process steps are as follows:

[0016] 1. Extraction of inclusion body protein After IPTG-induced expression, the bacteria were collected by centrifugation, the bacteria were ultrasonically broken, and refrigerated at high speed to obtain chicken Escherichia coli outer membrane protein inclusion body protein;

[0017] 2. Purification of inclusion body protein According to a certain mass-volume ratio, the inclusion body was resuspended in washing solution 1, washing solution 2 and washing solution 3 for washing, and finally the protein was precipitated by SLS, and the precipitate was collected by high-speed refrigerated centrifugation;

[0018] 3. Dissolution and renaturation of inclusion body protein. Resuspend the precipitate with denaturant according to...

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Abstract

The invention belongs to the technical field of protein purification, and relates to the field of downstream protein purification of biological medicine and bioengineering, and discloses a method for purifying a regrouped chicken colibacillosis outer membrane protein inclusion body and carrying out renaturation by utilizing high-efficiency renaturation liquid. By adopting a three-step washing purification technological method of the inclusion body proteins, mycoproteins can be well separated from the inclusion body, the renaturation rate of the proteins can be greatly improved by virtue of a renaturation solution and a dropwise feeding one-step renaturation technological method, the renaturation process and needed equipment can be simplified, the yield rate of the target protein can be greatly increased, and the production cost of the regrouped chicken colibacillosis outer membrane protein can be reduced. The regrouped chicken colibacillosis outer membrane protein purification method is simple and easy, and the column-passing treatment is not needed.

Description

technical field [0001] The invention relates to the field of purification of downstream proteins of biomedicine and bioengineering, and more specifically relates to a process for purifying inclusion bodies of recombinant chicken Escherichia coli outer membrane proteins and a high-efficiency refolding solution for refolding them. Background technique [0002] The outer membrane protein of Gram-negative bacteria plays an important role in the resistance of bacteria to harmful substances or the environment. It can protect cells from or reduce the influence of many harmful substances, such as proteases, antibiotics, toxins and phages, and maintain Gram Bacteria-negative bacteria play an important role in the survival of high osmotic pressure, acid and other stress conditions. In addition, non-specific ion channels formed by outer membrane proteins also play a key role in the absorption of nutrients and the excretion of metabolic wastes of Gram-negative bacteria. Outer membrane ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/245C07K1/14C07K1/113
CPCC07K14/245
Inventor 王连民刘珂飞孙珊珊
Owner TIANJIN SHENGJI GRP CO LTD
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