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Method of efficiently and conveniently purifying proteins

A protein and target protein technology, applied in the field of bioengineering, can solve problems such as non-specific cleavage of target protein, damage to target protein, and limited application

Active Publication Date: 2020-01-21
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commonly used tag removal methods include the use of proteases such as factor Xa, TEV protease, and rhinovirus 3C protease [14–16] , but proteases require additional purification steps to remove, and these purification steps will cause certain damage to the target protein, such as dissociation reactions that may lead to non-specific cleavage of the target protein or leave a small number of "residue" amino acids on the target protein
The high cost of using a large number of enzymes to remove protein tags severely limits their applications

Method used

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  • Method of efficiently and conveniently purifying proteins
  • Method of efficiently and conveniently purifying proteins
  • Method of efficiently and conveniently purifying proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Purification of enhanced green fluorescent protein eGFP

[0051] (1) The plasmid pET-28a-cipA-dnaB-eGFP was constructed, and the genes cipA (NCBI: CAE138691), dnaB (PDB: 1MI8_A) and eGFP (NCBI: AHK23750.1) were synthesized by OE-PCR. Ligate cipA and eGFP to the 5' and 3' ends of dnaB, respectively. cipA-dnaB-eGFP was cloned into pET-28a vector by Gibson assembly. Subsequently, the PCR product was transformed into E. coli XL10-Gold, and the plasmid was verified by DNA sequencing.

[0052] (2) Express the protein CipA-DnaB-eGFP, introduce the plasmid pET-28a-cipA-dnaB-eGFP into Escherichia coli BL21(DE3), pick a single colony and inoculate it in 5mL LB medium, add 50μg / mL Kana Seed solution was obtained by culturing overnight at 37°C. The next day, the seed solution was inoculated in 200mL fresh TB medium, and cultured at 37°C for about 2h, so that the OD 600 When it reaches 0.5-0.8, add 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) to the culture medium, and culture at...

Embodiment 2

[0058] Purification of β-galactosidase (β-Gal)

[0059] (1) The plasmid pET-28α-cipA-dnaB-lacZ (pET-28α-CIZ) was constructed, and the genes cipA (NCBI: CAE138691), dnaB (PDB: 1MI8_A) and lacZ (NCBI: 945006) were synthesized by OE-PCR . Then, cipA and lacZ were ligated to the 5' and 3' ends of dnaB by OE-PCR, respectively. The cipA-dnaB-lacZ fragment was then cloned into the pET-28a vector by Gibson assembly. Subsequently, the PCR product was transformed into Escherichia coli XL10-Gold, and the recombinant plasmid was verified by sequencing.

[0060] (2) Express the fusion protein CipA-DnaB-β-Gal, transform pET-28α-CIZ into Escherichia coli BL21(DE3), and inoculate a single colony in 5 mL LB medium, add 50 μg / mL kanamycin , inoculate the culture solution in 200 mL of fresh TB medium after culturing overnight at 37 °С. OD 600 When it reaches 0.5-0.8, add 0.5mM isopropyl-β-D-thiogalactoside (IPTG) and incubate at 30°C for 10h. In order to prevent the acidification of the me...

Embodiment 3

[0069] Purification of Maltose Binding Protein (MBP)

[0070] (1) The plasmid pET-28α-cipA-dnaB-MBP (pET-28α-CIM) was constructed, and the genes cipA (NCBI: CAE138691), dnaB (PDB: 1MI8_A) and MBP (Gene ID: 4155 ). Then, cipA and MBP were ligated to the 5' and 3' ends of dnaB by OE-PCR, respectively. The cipA-dnaB-MBP fragment was then cloned into the pET-28a vector by Gibson assembly. Subsequently, the PCR product was transformed into Escherichia coli XL10-Gold, and the recombinant plasmid was verified by sequencing.

[0071] (2) Express the fusion protein CipA-DnaB-MBP, transform pET-28α-CIM into Escherichia coli BL21 (DE3), and inoculate a single colony in 5 mL LB medium, add 50 μg / mL kanamycin, in After culturing at 37°C overnight, the culture solution was inoculated into 200 mL of fresh TB medium. OD 600 When it reaches 0.5-0.8, add 0.5mM isopropyl-β-D-thiogalactoside (IPTG) and incubate at 30°C for 10h. In order to prevent the acidification of the medium in the medi...

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Abstract

The invention provides a convenient and efficient purification method only requiring centrifugation and cracking steps. Traditional technologies of using column chromatography for purifying recombinant proteins are high in production cost and cumbersome in step; and by adopting non-chromatographic purification methods based on affinity tags, costs can be reduced, the yield of target proteins is increased, but the cost of tag removal is high. In order to solve the problems, the convenient and efficient purification method only requiring the centrifugation and cracking steps is provided. According to the convenient and efficient purification method, an inclusion-body protein CipA and an intein protein (Synechocystis sp.PCC6803 DnaB, Ssp DnaB) are adopted, a self-assembly function of the CipAand a self-cracking function of the DnaB under weak-acid conditions are utilized for achieving protein purification. A target protein can be any protein needing to be expressed and purified, and thepurity of the purified target protein can achieve 95% or above. Besides, according to the convenient and efficient purification method, a connection peptide between the CipA and the DnaB is also optimized, therefore, the cracking efficiency of the DnaB is further improved, and meanwhile influences of the CipA on activities of the target protein are reduced. The convenient and efficient purification method only requiring the centrifugation and cracking steps has the potential of being applied to industrial production of enzymes. Keywords include the protein purification method, the inclusion-body protein CipA, and the self-cracking protein DnaB.

Description

technical field [0001] The invention relates to a plasmid pET-28a-cipA-dnaB-eGFP expressing inclusion body protein CipA, a self-cleaved intein and target protein eGFP. The invention also relates to a method for constructing the plasmid and a method for purifying the target protein, belonging to the technical field of bioengineering. Background technique [0002] With the development of molecular biology, cell engineering and fermentation engineering, the expression efficiency of recombinant proteins has been significantly improved. Despite this success, developing a large-scale, facile, and efficient method for protein purification remains a formidable challenge. In enzyme-catalyzed processes, enzyme purity is an essential step in reducing the cost of producing the desired product. Many studies mention purification of target protein using different methods such as affinity purification [1,2] , using a self-aggregating purification tag [3-5] and self-cleavage tag (intein)...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/62C12N1/21C07K14/435C12R1/19
CPCC07K14/43595C07K2319/00
Inventor 陈振娅霍毅欣赵璐瑶
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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