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Target protein-intein-magnetosome fusion gene, intelligent bacterial expression system constructed by target protein-intein-magnetosome fusion geneand used for automatically purifying product, and preparation method of intelligent bacterial expression system

A technology of fusion gene and expression system, applied in the field of genetic engineering, can solve the problems of protein yield reduction, protein destruction, function destruction, etc., and achieve the effects of low cell fluid viscosity, easy purification, and low production cost

Inactive Publication Date: 2021-11-02
张金菊 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In actual production, it is necessary to explore the process conditions of each step, and each condition exploration will inevitably lead to a decrease in protein yield, activity, or function destruction
In other words, the more complicated the steps, the more serious the damage to the protein will be
Reports show that in the large-scale production of industrial and pharmaceutical proteins, the cost of subsequent protein purification is as high as 80% of the cost of the entire process

Method used

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  • Target protein-intein-magnetosome fusion gene, intelligent bacterial expression system constructed by target protein-intein-magnetosome fusion geneand used for automatically purifying product, and preparation method of intelligent bacterial expression system
  • Target protein-intein-magnetosome fusion gene, intelligent bacterial expression system constructed by target protein-intein-magnetosome fusion geneand used for automatically purifying product, and preparation method of intelligent bacterial expression system
  • Target protein-intein-magnetosome fusion gene, intelligent bacterial expression system constructed by target protein-intein-magnetosome fusion geneand used for automatically purifying product, and preparation method of intelligent bacterial expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] The construction of embodiment 1 recombinant suicide plasmid

[0089] 1. Use the primers in Table 1 and high-fidelity pfu enzyme to PCR amplify the mamC upstream arm gene, anti-tetrabromobisphenol A nanobody gene, intein gene, mamC gene and mamC downstream arm gene. The PCR system and conditions are as follows:

[0090] PCR system (50μl):

[0091]

[0092] PCR conditions (denaturation → annealing → extension: 30 cycles):

[0093]

[0094] Table 1 Primers used in PCR amplification

[0095]

[0096]

[0097] Note: The underline is the restriction site sequence.

[0098] 2. According to the instructions of the agarose gel DNA recovery kit of Tiangen Biochemical Technology (Beijing) Co., Ltd., the PCR amplification product in step 1 was gel-cut and recovered;

[0099] 3. Use fusion PCR technology to carry out 3-step fusion PCR amplification of each gene fragment recovered in step 2:

[0100] Firstly, fusion gene 1 is obtained by fusion amplification of nano...

Embodiment 2

[0131] Example 2 Screening of recombinant expression strain 1

[0132] The recombinant plasmid pKuTBInCd in the donor strain E. coli S17-1 in Example 1 was transformed into the recipient strain M. gryphiswaldensense MSR-1 by parental conjugation experiment. Specifically: 1) S17-1 single colony was inoculated in 4ml liquid LB, cultured overnight at 37°C, 200rpm; 2) S17-1 was inoculated in 10% antibiotic-free liquid LB (200μl S17-1+2ml LB ), 30°C, 150rpm, cultured for 3h; 3) MSR-1 was activated twice in sodium lactate medium, and cultured for the third time to OD 565 0.8-1.0; 4) Mix 300 μl of cultured S17-1 in 2) and 1 ml of MSR-1 in 3), centrifuge at 12,000 rpm for 1 min, and discard the supernatant in an ultra-clean bench; 5) Use 1 ml of glutamine Wash the bacteria with sodium glutamate selective medium, centrifuge at 12,000rpm for 1min, discard the supernatant in an ultra-clean workbench, and repeat once; 6) Resuspend the bacteria in 5) with 50 μl of sodium glutamate selecti...

Embodiment 3

[0147] The construction of embodiment 3 recombination shuttle plasmids

[0148] 1. Utilize the primers in Table 2 and the high-fidelity pfu enzyme to amplify the lyase gene and the nuclease gene by PCR respectively. The PCR system and conditions are as follows:

[0149] PCR system (50μl):

[0150]

[0151]

[0152] PCR conditions (denaturation → annealing → extension: 30 cycles):

[0153]

[0154] Table 2 Primers used in PCR amplification

[0155]

[0156]

[0157] Note: The underline is the restriction site sequence.

[0158] 2. According to the instructions of the agarose gel DNA recovery kit of Tiangen Biochemical Technology (Beijing) Co., Ltd., the PCR amplification product in step 1 was gel-cut and recovered;

[0159] 3. Use fusion PCR technology to add signal peptide to the nuclease gene recovered in step 2 and add a promoter to the lyase gene. The signal peptide sequence and promoter sequence are shown in Table 2:

[0160] The reaction system and con...

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Abstract

The invention provides a target protein-intein-magnetosome fusion protein, an intelligent bacterium constructed by using the target protein-intein-magnetosome fusion protein and used for automatically purifying a product and a preparation method of the intelligent bacterial expression system, and the fusion protein is formed by directly connecting a target protein, intein and magnetosome membrane protein in series or operably connecting the target protein, the intein and the magnetosome membrane protein through a flexible Linker; the invention also provides a target protein-intein-magnetosome fusion gene. The fusion gene comprises an upstream sequence of a magnetosome membrane protein gene, a nano antibody gene, an intein gene, the magnetosome membrane protein gene and a downstream sequence of the magnetosome membrane protein gene; and the invention also provides an intelligent bacterium constructed by using the target protein-intein-magnetosome fusion gene and capable of automatically purifying a product . Compared with a traditional prokaryote expression system, the intelligent bacterium has the advantages of being mild in target protein purification condition, free of additional reagents, low in cost, easy to operate, high in purity and small in influence on the yield and the like of the target protein.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an intelligent bacterial expression system and a preparation method for automatic purification of target protein-intein-magnetosome fusion gene and its constructed product. Background technique [0002] Bacteria are characterized by short passage times, high-density production, and simple media components, making them the first choice for simple heterologous gene expression without transcriptional or post-translational modifications. In addition to having the general characteristics of bacteria, Escherichia coli is the most favored in laboratory and industrial production because of its clearest genetic background and easy operation. The purpose of heterologous protein expression is to obtain high-yield functional protein products, which requires first breaking the cells to release the target protein, and then screening the target protein from the cell debris. There...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00C12N1/21C12N15/55C12N15/60C12N15/74C07K1/14C12R1/01
CPCC07K14/00C12N9/88C12N9/22C12N15/74C07K1/14
Inventor 张金菊王红光
Owner 张金菊
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