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Recombinant Escherichia coli capable of realizing soluble expression of linoleate isomerase and application of recombinant Escherichia coli

A technology of recombinant Escherichia coli and linoleic acid isomerase, which is applied in the field of bioengineering and can solve the problems of low soluble expression level and the like

Pending Publication Date: 2018-10-16
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to address the defects in the prior art, especially the low level of soluble expression of linoleic acid isomerase PAI in Escherichia coli, by constructing new recombinant Escherichia coli and improving its induction culture method to improve Soluble expression level of PAI protein in Escherichia coli in order to enhance its ability to produce t10,c12-CLA

Method used

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  • Recombinant Escherichia coli capable of realizing soluble expression of linoleate isomerase and application of recombinant Escherichia coli
  • Recombinant Escherichia coli capable of realizing soluble expression of linoleate isomerase and application of recombinant Escherichia coli
  • Recombinant Escherichia coli capable of realizing soluble expression of linoleate isomerase and application of recombinant Escherichia coli

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Effect test

Embodiment 1

[0029] Embodiment 1: Construction method of recombinant Escherichia coli BL21 (DE3) (pET24a-Mpai)

[0030]Using the genome of Escherichia coli K12 (NC_000913.3) containing the gene malE of the maltose-binding protein MBP as a template, the whole malE gene was synthesized and connected to the plasmid pUC57, and pUC57- malE, excise the malE gene and recover by cutting the gel, and the same expression vector pET24a-pai containing the pai gene (Genbank: AX062088.1) (for the construction method, refer to "Linoleic acid isomerase gene in oleaginous fungi) Heterologous expression and biosynthesis of products" (Zhang Baixi)); after ligation reaction at 16°C overnight, the malE gene was inserted into the upstream of the pai gene of the corresponding expression vector, and the fusion protein expression vector pET24a-Mpai was constructed ( figure 1 ). Transform E.coli BL21 (DE3) competent and spread on LB plates containing 50 μg / mL kanamycin, and identify the positive clones after trans...

Embodiment 2

[0031] Embodiment 2: Product analysis of recombinant Escherichia coli BL21 (DE3) (pET24a-Mpai)

[0032] Pick a single colony of the recombinant strain obtained in Example 1, inoculate it in 5 mL of LB medium containing 50 μg / mL kanamycin, cultivate overnight at 37° C. with shaking at 200 r / min, and then inoculate it at a ratio of 2% (v / v). Transfer to 50mL LB medium containing 50μg / mL kanamycin, culture at 37°C, 200r / min shaking for 3-4h to OD 600 The value is 0.6-0.8, add IPTG to a final concentration of 1mmol / L, and culture with shaking at 20°C for 24h.

[0033] Collect the bacteria by centrifugation at 4°C and 10,000r / min, and resuspend them in 100mmol / L Tris-HCl buffer (pH7.4), sonicate the bacteria and centrifuge at 4°C and 6,000r / min for 10min, and collect the supernatant It is the whole-cell protein, take 10 μL whole-cell protein, add 10 μL 1× protein loading buffer, pipette and beat to mix evenly, then bathe in boiling water for 10 min, and use SDS-PAGE electrophoresi...

Embodiment 3

[0035] Example 3: Optimization of expression conditions induced by recombinant Escherichia coli BL21 (DE3) (pET24a-Mpai)

[0036] With the Escherichia coli BL21 (DE3) (pET24a-Mpai) that embodiment 1 obtains as object, with the LB culture medium of embodiment 2 as the basis, consider the temperature of induced expression, inducer concentration and induction time to fusion protein MBP-PAI effect to further increase the expression level.

[0037] (1) Induction temperature optimization

[0038] Induction temperature is very important to the growth of bacteria and protein expression. In order to explore the optimal induction temperature, the recombinant protein MBP-PAI was expressed under different induction temperature conditions.

[0039] Pick a single bacterium colony of the recombinant strain obtained in Example 1, inoculate in 5 mL of LB medium containing 50 μg / mL kanamycin, cultivate overnight at 37° C., and transfer to 50 mL of In LB medium containing 50 μg / mL kanamycin, c...

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Abstract

The invention discloses recombinant Escherichia coli capable of realizing soluble expression of linoleate isomerase and application of the recombinant Escherichia coli, and belongs to the technical field of bioengineering. The recombinant Escherichia coli is constructed by the following steps: inserting genes mal E of a coded maltose-binding protein MBP into the upstream of genes pai of linoleateisomerase PAI, and constructing the recombinant Escherichia coli containing an MBP-PAI fusion expression vector. The expression quantity of the PAI of the obtained recombinant Escherichia coli BL21 (DE3) (pET24a-Mpai) is far higher than that of other strains recorded in the prior art, and most of the recombinant proteins MBP-PAI exist in a soluble protein form. Therefore, the recombinant Escherichia coli disclosed by the invention is capable of realizing high-efficiency soluble expression of the MBP-PAI, and the problem of excessive inclusion bodies is solved.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli for soluble expression of linoleic acid isomerase and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Trans 10, cis 12-conjugated linoleic acid (t10,c12-CLA) is an octadecadienoic acid with two conjugated double bonds, as one of the most important active monomers of conjugated linoleic acid , t10,c12-CLA has a variety of physiological functions, such as reducing body fat accumulation, preventing atherosclerosis, regulating immunity, anti-tumor, etc. Therefore, this fatty acid has become a hot research topic at present. Has great development prospects. [0003] Linoleic acid isomerase (PAI) is an isomerase from Bacillus acnes, which can efficiently catalyze the conversion of linoleic acid into t10,c12-CLA. "Heterologous Expression of Linoleic Acid Isomerase Gene in Oleaginous Fungi and Biosynthesis of Products" Constructed recombinant Esch...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/61C12N15/70C12N9/90C12R1/19
CPCC12N9/90C12N15/70C12Y502/01005
Inventor 陈卫张白曦黄昕畑陈海琴赵建新张灏
Owner JIANGNAN UNIV
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