Escherichia coli expression system containing manganese ion recombinant protein, and application method thereof

A technology of Escherichia coli and expression system, applied in microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of complex renaturation process, high cost, inactivity, etc., and achieve simple separation and purification process, The effect of low production cost and increased vitality

Active Publication Date: 2018-12-14
WUHAN KANGFUDE BIOTECH CO LTD
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Problems solved by technology

[0009] Aiming at the problems in the prior art that inactive inclusion bodies are often obtained when expressing recombinant proteins containing manganese ions in Escherichia coli, and the renaturation process is complicated and the cost is high, one of the purposes of the present invention is to provide recombinant proteins containing manganese ions. Bacillus expression system, which contains Escherichia coli strains and corresponding plasmids, can produce soluble and active enzyme proteins containing manganese ions using this system

Method used

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  • Escherichia coli expression system containing manganese ion recombinant protein, and application method thereof
  • Escherichia coli expression system containing manganese ion recombinant protein, and application method thereof
  • Escherichia coli expression system containing manganese ion recombinant protein, and application method thereof

Examples

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Embodiment 1

[0094] Embodiment 1 Design of the enzyme protein gene expression cassette containing manganese ions

[0095] The schematic diagram of the universal vector for the co-expression of the Escherichia coli molecular chaperone gene, the enzyme protein gene containing manganese ions and the channel protein gene related to the pumping of manganese ions constructed in this example is as follows figure 1 shown. Among them, promoter 1 and promoter 2 are medium-strength promoters or weak promoters that can promote the expression of molecular chaperone genes and manganese ion channel-related protein genes in Escherichia coli, such as the P43 promoter sequence (SEQ ID NO.6, from subtilis), M1-93 promoter (SEQ ID NO. 7), araBAD promoter (SEQ ID NO. 8) and Lac promoter (SEQ ID NO. 9). Promoter 1 and promoter 2 can be the same promoter, or different promoters can be selected. The molecular chaperone gene is one or more of dnaK-dnaJ-grpE, groES-groEL, groES-groEL-tig or tig. The manganese io...

Embodiment 2

[0096] Example 2 Construction of oxalate decarboxylase A2 gene expression plasmid pET28a-A2

[0097] Using the A2 gene (SEQ ID NO.1) synthesized from the whole gene as a template, design a primer pair F1 / R1 to amplify the gene, and perform gel recovery and purification on the amplified product. The method refers to the commercially available DNA mini-purification kit. According to the method described in the specification, the DNA fragment 1 (ie, the A2 gene fragment) is finally obtained. The PCR system is: 10×PCR Buffer 5μL, 2mM dNTP 5μL, 25mMMgSO 45 μL, 1.5 μL each of 10 μM primer F / R, 0.5 μL template DNA, 1 μL KOD-Plus-Neo, 32.5 μL ddH2O; PCR reaction conditions are as follows: 94°C for 3 min, 30 cycles (98°C for 10s, 60°C for 30s, 68°C for 35s ), 68°C for 5 minutes, and 4°C for 10 minutes; the PCR system in the description of the following vector construction is consistent with the above description, and will not be repeated below. The PCR reaction conditions are slightly...

Embodiment 3

[0104] Example 3 Molecular chaperone overexpression strain construction

[0105] Transform five kinds of commercial chaperone plasmids pG-KJE8, pGro7, pKJE7, pG-Tf2 and pTf16 into commercial strains, screen positive clones on the LB solid medium plate containing 20 μg / ml, and use BL21(DE3) and Origami2 (DE3) expression strains were used as host bacteria to construct molecular chaperone overexpression strains. Competent cell preparation and E. coli transformation refer to "Molecular Cloning Experiment Guide (3rd Edition)" to obtain a series of strains: pG-KJE8 / BL21(DE3), pGro7 / BL21(DE3), pKJE7 / BL21(DE3), pG- Tf2 / BL21(DE3), pTf16 / BL21(DE3), pG-KJE8 / Origami2(DE3), pGro7 / Origami2(DE3), pKJE7 / Origami2(DE3), pG-Tf2 / Origami2(DE3) and pTf16 / Origami2( DE3).

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Abstract

The invention discloses an Escherichia coli expression system containing a manganese ion recombinant protein, and an application method thereof. A recombinant expression plasmid in the Escherichia coli expression system is one or a combination of the following cases: (1) an Escherichia coli molecular chaperone gene and an manganese ion-containing enzyme protein gene; (2) an E. coli chaperone geneAn enzyme protein gene containing manganese ions, and a channel-associated protein gene for overexpressing or inhibiting manganese ions; and (3) the Escherichia coli molecular chaperone gene, the manganese ion-containing enzyme protein gene, and a concentration-associated protein gene for affecting intracellular manganese ions. The Escherichia coli expression system is constructed and optimized torealize the efficient, soluble and active expression of the manganese ion-containing enzyme protein; and compared with conventional methods, the method has the advantages of high efficiency, simple purification process, low cost, and facilitation of the industrial production and application of manganese ion-containing enzymes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an Escherichia coli expression system and an application method of a manganese ion-containing recombinant protein. Background technique [0002] Escherichia coli is the first host bacteria used for recombinant protein production. It not only has clear genetic background, simple cultivation operation, high transformation and transduction efficiency, fast growth and reproduction, low cost, and can produce target proteins quickly and on a large scale. , has been the most widely used expression system in genetic engineering (Rong Jingjing et al. Research progress in Escherichia coli expression system. Pharmaceutical Biotechnology, 2005,12(6):416-420; Xie Tingbo, Research progress in Escherichia coli expression system. Yangtze River University Journal (Natural Science Edition), 2008,5(3):77-83). Studies in recent years have shown that the E. coli expression vectors widely use...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12N9/88C12N9/02C12N9/78C12R1/19
CPCC12N9/0008C12N9/0089C12N9/78C12N9/88C12N15/70C12Y102/03004C12Y115/01001C12Y305/03001C12Y401/01002
Inventor 汪小锋吴玉峰汪卫刘艳红陈火晴
Owner WUHAN KANGFUDE BIOTECH CO LTD
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