Method for separating and purifying fusion protein containing chitin binding domain

A technique of combining structural domains and separation and purification, which is applied in the field of fusion proteins and can solve problems such as unsatisfactory adsorption effects

Inactive Publication Date: 2014-08-13
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, natural chitin is a straight-chain polymer composed of N-acetylglucosamine with β-1,4 glycosidic bonds. It is relatively stable and insoluble in water, dilute alkali, dilute acid and most organic solvents. Directly applied to the separation and purification of fusion proteins containing chitin-binding domains, the adsorption effect is often not ideal

Method used

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  • Method for separating and purifying fusion protein containing chitin binding domain
  • Method for separating and purifying fusion protein containing chitin binding domain
  • Method for separating and purifying fusion protein containing chitin binding domain

Examples

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Effect test

Embodiment 1

[0015] Example 1 Preparation of Partially Deacetylated Chitin Derivative Column Material

[0016] Take 30g of chitin (Aladdin Reagent (Shanghai) Co., Ltd.) in a 1000mL beaker, add 200mL of double distilled water to soak overnight; pour off the double distilled water, add 500mL of 2mol / L HCl to soak for 2h, suction filter, and then wash with water until neutral Add 500mL lmol / L NaOH to soak, and boil the beaker in a 100℃ water bath for 1h, suction filter, and then wash until neutral; add 500mL0.01mol / L NaOH to soak, put the beaker in a 50℃ water bath Boil in water for 1 hour, filter with suction, wash with water until neutral; add 500mL of 0.01mol / LCH 3 COOH, boil the beaker in a 50°C water bath for 0.5h, filter with suction, and wash with water until neutral. Finally, place the treated chitin in a vacuum desiccator for vacuum drying treatment. The temperature of the vacuum desiccator should not exceed 80°C. After complete drying, put the chitin in a mortar for grinding, store...

Embodiment 2

[0017] Example 2 Design and Cloning of a Fusion Protein Containing a Chitin Binding Domain (CBD)

[0018] According to the amino acid sequence of the lunasin polypeptide, the gene of the lunasin polypeptide was designed using OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER) and Gene Designer (http: / / www.DNA20.com) software and codons preferred by Escherichia coli coding sequence, and added Sap I and Pst I restriction sites on both sides. The lunasin polypeptide coding gene sequence containing Sap I and Pst I restriction sites on both sides was obtained by overlap extension PCR method, and TA-cloned into pMD TM In the 19-T Simple Vector (product of TaKaRa Biotech (Dalian) Company), Sap I and Pst I were double digested, and then subcloned into the pTWIN1 vector (product of New England Biolabs Company) to obtain the recombinant plasmid pTWIN1-1un ( figure 1 ), wherein the fusion expression gene sequentially includes chitin binding domain (Chitin Binding Domain, CBD) gene, Ssp DnaB in...

Embodiment 3

[0019] Example 3 Escherichia coli shake flask expression of CBD-Ssp DnaB intein-lunasin protein and product identification

[0020] Pick a single colony of the positive clone pTWIN1-1un / E.coli BL21(DE3) into 50ml LBA (LB medium with 25μl 20% (w / v) Amp) liquid seed medium, and shake at 220rpm at 37°C for 12h. Seed medium was inoculated in 200ml expression medium (containing 80μl trace element medium, 100μl 20% (w / v) Amp) with 1% (v / v) inoculum amount (1L expression medium contained 25g glycerol; yeast powder 15g ; Peptone 15g; (NH 4 ) 3 PO 4 ·3H 2 O4g; KH 2 PO 4 8g;K 2 HPO 4 ·3H 2 O7g; MgSO 4 ·7H 2 O1g; 1L trace element medium contains HCl5ml; CaCl 2 2H 2 O9g; CoCl 2 ·6H 2 O6g; CuCl 2 2H 2 O2.125g; FeCl 3 ·6H 2 O135g; H 3 BO 3 0.75g; MnCl 2 4H 2 O5g; Na 2 MoO 4 2H 2 O12g; ZnO5g), 37 ° C, 220rpm shaking culture to OD 600nm=0.6-0.7, at this time, 2ml of 20% (w / v) lactose was added to a final concentration of 0.2% (w / v), and the induction expression was c...

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Abstract

The invention provides application of a partially deacetylated chitin derivative in separation and purification of fusion protein containing a chitin binding domain. The principle used in the invention is that chitin undergoes deacetylation through NaOH alkali lye heating and then acetic acid treatment is carried out to acetylate the deacetylated chitin so as to obtain the partially deacetylated chitin derivative. The partially deacetylated chitin derivative is applied to separation and purification of fusion protein containing the chitin binding domain and has a better improved specific adsorption effect on fusion protein containing the chitin binding domain.

Description

technical field [0001] The invention belongs to the field of downstream separation and purification of genetic engineering products, and relates to the application of partially deacetylated chitin derivatives to specific adsorption, separation and purification of fusion proteins containing chitin binding domains (Chitin Binding Domain, CBD). Background technique [0002] When using genetic engineering technology to express some exogenous polypeptides or small molecular proteins, the expression products are often degraded by proteases in the host cells, resulting in a greatly reduced expression. In order to effectively solve this problem, fusion expression technology is widely used at present. The so-called fusion expression refers to splicing exogenous small molecular protein (polypeptide) downstream of the fusion partner (tag) for expression through gene recombination technology, and then releases the target small molecular protein (polypeptide) through enzymatic or chemica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/22
Inventor 谭树华张怡
Owner CHINA PHARM UNIV
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