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Method for promoting efficient water-soluble expression of arginine deiminase

An arginine deiminase and water-soluble technology, which is applied in the biological field to achieve the effect of promoting high-efficiency water-soluble expression and overcoming the insoluble expression of arginine deiminase

Active Publication Date: 2015-04-15
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In Escherichia coli, the water-soluble yield of recombinant proteins is mainly improved through strategies such as molecular chaperone co-expression, secretory expression, fusion expression and optimization of culture conditions. However, these optimization strategies cannot effectively solve the problems encountered in the expression and purification of each protein. For some special proteins, it is still necessary to explore some new and more effective optimization strategies to help them express their activity
After searching, on the basis of the co-expression of molecular chaperones, there is no report on the method of promoting the efficient water-soluble expression of arginine deiminase by adding substrates L-arginine and D-glucose during the cultivation of E. coli

Method used

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  • Method for promoting efficient water-soluble expression of arginine deiminase
  • Method for promoting efficient water-soluble expression of arginine deiminase
  • Method for promoting efficient water-soluble expression of arginine deiminase

Examples

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Effect test

Embodiment 1

[0026] Example 1 Arginine deiminase derived from Pseudomonas putida was constructed as a recombinant expression vector and expressed heterologously in Escherichia coli.

[0027] (1) Arginine deiminase (GenBank accession no.P41142) derived from Pseudomonas putida was used according to the codon frequency table of the expression host E.coliBL21(DE3), using the "codon random distribution" Optimize the strategy to fully artificially synthesize the ADI gene. Then, using the PCR method, the corresponding restriction endonucleases NcoI and HindIII were cloned into the upstream and downstream of the ADI gene, and connected to the pET-30a-c(+) vector containing the 6His tag to construct the recombinant expression vector pET30a- ADI, and co-transformation with the pGro7 plasmid encoding the molecular chaperone GroEL / GroES into E.coliBL21(DE3) to obtain engineering bacteria containing double plasmids (pET30a-ADI and pGro7).

[0028] The "codon random distribution" strategy refers to weigh...

Embodiment 2

[0042] The method is the same as in Example 1, except that the concentration of D-glucose in step (3) is 1g / L, the final concentration of L-arabinose in step (3) is 0.2g / L, and the concentration of L-arabinose in step (4) The concentration of arginine was 1 g / L.

Embodiment 3

[0044] The method is the same as in Example 1, except that the concentration of D-glucose in step (3) is 10g / L, the final concentration of L-arabinose in step (3) is 0.8g / L, and the concentration of L-arabinose in step (4) Arginine concentration is 10g / L.

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Abstract

The invention discloses a method for promoting efficient water-soluble expression of arginine deiminase (ADI). The method comprises the following steps: connecting ADI gene derived from Pseudomonas putida with a pET-30a-c(+) carrier to construct a recombinant expression plasmid pET30a-ADI, and cotransforming the pET30a-ADI and pGro7 plasmid into E.coliBL21(DE3) to obtain double-plasmid-containing engineering bacteria; inoculating the engineering bacteria into an LB (Langmuir-Blodgett) culture medium containing 1-10 g / L D-glucose, adding L-arabinose, and culturing; and when the bacteria are cultured to a logarithmic phase, adding 1-10 g / L L-arginine and 0.1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside), and carrying out induced culture at 16 DEG C under the condition of 180 rpm. The method disclosed by the invention can obviously enhance the water-soluble yield of the recombinant ADI protein, enhances the activity of the crude enzyme solution by 15 times, effectively overcomes the defect of difficulty in soluble expression of the recombinant ADI, provides technical support for application of industrialized large-scale production of citrulline and the like, and also provides a new idea for expression of other recombinant proteins.

Description

technical field [0001] The invention relates to a method for promoting the water-soluble expression of heterologous proteins in Escherichia coli, in particular to a method for promoting the efficient water-soluble expression of arginine deiminase in Escherichia coli by adding substrates, and belongs to the field of biotechnology . Background technique [0002] As a heterologous expression host, Escherichia coli has become the main method to obtain a large amount of target protein because of its simple culture conditions, short passage time, and mature genetic manipulation system. However, overexpression in the E. coli system often leads to protein misfolding, aggregation, degradation, low activity, and existence in the form of inclusion bodies, which limits its related research and application. [0003] Arginine deiminase (Arginine deiminase, EC3.5.3.6; ADI) catalyzes the hydrolysis of L-arginine into L-citrulline and ammonia, and ornithine carbamoyltransferase, carbamoyl p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/78C12N15/70C12R1/40
Inventor 李越中王颖
Owner SHANDONG UNIV
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