Escherichia coli expression system and application method of recombinant protein containing manganese ions

A technology of Escherichia coli and expression system, applied in the direction of microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of high cost, complex renaturation process, inactivity, etc., and achieve the effect of improving vitality

Active Publication Date: 2021-06-18
WUHAN KANGFUDE BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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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 and application method of recombinant protein containing manganese ions
  • Escherichia coli expression system and application method of recombinant protein containing manganese ions
  • Escherichia coli expression system and application method of recombinant protein containing manganese ions

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 of recombinant protein containing manganese ions and an application method thereof. The recombinant expression plasmid in the Escherichia coli expression system is one of the following situations or a combination thereof: (1) at least comprising an Escherichia coli molecular chaperone gene and an enzyme protein gene containing manganese ions; (2) comprising an Escherichia coli molecular chaperone gene , enzyme protein genes containing manganese ions, and overexpression or inhibition of manganese ion channel-related protein genes; (3) including Escherichia coli molecular chaperone genes, enzyme protein genes containing manganese ions, and protein genes affecting intracellular manganese ion concentration . Through the construction and optimization of the Escherichia coli expression system, efficient soluble and active expression of the manganese ion-containing enzyme protein can be realized. Compared with the traditional method, the efficiency is high, the purification process is simple, and the cost is low, which is beneficial to the production of the manganese ion-containing enzyme. Industrial production and application.

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 Patents(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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