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Application of cellulose binding domain in secretory expression system of corynebacteria

A coryneform bacteria, secreted expression technology, applied in the field of rapid purification of recombinant proteins secreted and expressed by coryneform bacteria, can solve the problems of increasing the cost of protein purification, high manufacturing costs, etc.

Inactive Publication Date: 2014-08-06
SHAOXING INST OF TECH COLLEGE OF ENG PEKING UNIV BIOENG CENT
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  • Abstract
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  • Claims
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Problems solved by technology

The high manufacturing cost of buffers and affinity resins used in these methods will increase the cost of protein purification, and in large-scale production applications, cost is often an important factor that must be considered

Method used

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  • Application of cellulose binding domain in secretory expression system of corynebacteria
  • Application of cellulose binding domain in secretory expression system of corynebacteria
  • Application of cellulose binding domain in secretory expression system of corynebacteria

Examples

Experimental program
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Effect test

Embodiment

[0038] Example: secreted expression of eGFP-CBD and its cellulose column purification

[0039] eGFP is a green fluorescent protein commonly used for molecular localization and protein labeling. This example takes eGFP as an example, through its secreted expression and purification by fusion with CBD in Corynebacterium glutamicum, so as to demonstrate the application of CBD in affinity chromatography.

[0040] Strains and Reagents

[0041]Escherichia coli E.coli DH5a, plasmid pMXS-IRES-GFP, Corynebacterium glutamicum C.glutamicum13032, expression vector pXMJ19-sp of Corynebacterium glutamicum are owned by our laboratory, DNA purification and recovery kit, plasmid extraction kit , Protein Marker was purchased from Tiangen Biological Co., Ltd.; T4 ligase, Taq polymerase and various restriction enzymes were purchased from NEB Company; nickel column and dialysis membrane were purchased from Sangon Bioengineering Co., Ltd.; microcrystalline cellulose filler Avicel PH-102NF (batch ...

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Abstract

The invention specifically relates to a method of using the cellulose binding domain of Trichoderma reesei as an affinity tag for secretory expression and purification of recombinant protein in a corynebacteria expression system. The method comprises the following steps: 1) optimizing and synthesizing a codon sequence coding the cellulose binding domain of Trichoderma reesei glucoside hydrolase I; 2) cloning the sequence synthesized in the step 1 to a corynebacteria secretory expression vector as a protein tag sequence; 3) inserting a target protein sequence into the tag vector obtained in the step 2; 4) transforming the recombinant vector obtained in the step 3 to corynebacteria for secretory expression of protein; and 5) with CBD as the affinity tag, purifying recombinant protein secreted by the recombinant bacterium by using a cellulose column. According to the method, fragmentation of thalli is not needed, a low-cost cellulose column filling material is directly used for protein purification of an induced medium, so production cost for protein is reduced.

Description

technical field [0001] The invention belongs to the technical field of protein expression and purification, and specifically relates to a method for quickly purifying recombinant proteins secreted and expressed by coryneform bacteria by using cellulose binding domains as affinity tags. The invention relates to the construction of a coryneform bacterium secretion expression vector with CBD as an affinity tag and the purification of the recombinant protein secreted by the coryneform bacterium by using a cellulose column. Background technique [0002] Corynebacteria are non-pathogenic, Gram-positive bacteria that are commonly used in the production of various commercially available amino acids and vitamins [1-4] . After years of mutation breeding, the improvement of protoplasts and expression vectors is expected to be used for large-scale protein production [5，6] . In 2006, Japan's Date et al. used Corynebacterium glutamicum to secrete and express active human epidermal grow...

Claims

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Application Information

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IPC IPC(8): C12N15/77C07K1/22
Inventor 赵志敬蒋欢胡广
Owner SHAOXING INST OF TECH COLLEGE OF ENG PEKING UNIV BIOENG CENT
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