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Method for efficient secretory expression of foreign proteins by use of bacillus

A bacillus, secretion expression technology, applied in the field of high-efficiency secretion and expression of foreign proteins, can solve the problems of slow folding of foreign proteins and low expression of foreign proteins, and achieve short fermentation cycle, change of lifestyle and low production cost Effect

Inactive Publication Date: 2017-10-24
CHENGDU MYTECH BIOTECH +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the Sec pathway has an internal regulatory mechanism that inhibits the secretion of incorrectly folded proteins, and foreign proteins generally fold slowly, and the folding process requires the participation of chaperone proteins, which are easily degraded by the Sec secretion pathway, which results in only a very small number of proteins that can utilize Sec. pathway for secretory expression, and the expression level of foreign proteins is relatively low

Method used

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  • Method for efficient secretory expression of foreign proteins by use of bacillus
  • Method for efficient secretory expression of foreign proteins by use of bacillus

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Embodiment 1

[0046] 1. Cut pBAV1K-T5-GFP by EcoR I and Apa I endonuclease ( http: / / www.addgene.org / Vector-database / ) plasmid was cloned by homologous recombination with the synthesized MCS fragment to obtain plasmid pBAV1K.

[0047] The endonuclease used in this experiment and subsequent experiments was the rapid endonuclease from Thermo Company, and the gel recovery kit (DE-02011) from Chengdu Fuji Biological Company was used for fragment recovery. The MCS fragment was synthesized by Jinweizhi Biotechnology Co., Ltd. The EsayGeno rapid recombination cloning kit (VI201-02) from Tiangen was used for source recombination, the E. coli strain was top10, the competent state was prepared by the KCM method, and the plasmid was extracted using the Universal Plasmid Small Extraction Kit (DE-01001 ).

[0048] 2. Design primers to amplify the pBAV1K fragment with synonymous mutation to delete the Nde I restriction site, connect the amplified fragments by homologous recombination cloning, and obtai...

Embodiment 2

[0051] 1. For the strain transformation method, see the hyperosmolarity transformation method (High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis, 1999). Take a newly streaked single colony of the strain to be transformed, inoculate it into about 3ml of GM (LB+0.5M sorbitol) medium, and culture overnight at 37°C and 180rpm. The next morning, inoculate into 50ml GM medium at a ratio of 1:100, incubate at 37°C and 180rpm, and when the OD reaches 0.85-0.95, place the bacterium on ice to pre-cool for 10 minutes; at 4°C, Centrifuge at 5000g for 5min to remove the supernatant, resuspend the cells with an equal volume of pre-cooled EM (0.5M sorbitol + 0.5M mannitol + 10% glycerin aqueous solution), and centrifuge again at 4°C at 5000g for 5min to remove the supernatant. Clean, repeat washing 4 times in total; add about 1 / 40 volume of EM resuspended bacteria to ensure that the concentration of the b...

Embodiment 3

[0053] Bacillus subtilis Z12 (((Bacillus subtilis Z12 in Latin), preserved in the General Microorganism Center (CGMCC) of the China Microbiological Culture Collection Management Committee (CGMCC), the preservation number is 12750, and the preservation date is July 11, 2016)) The colony is round , is white or light yellow, opaque, showing roughness, wrinkles, and irregular edges. The growth process of Z12 needs oxygen, the optimum pH for growth is 7.0-8.5, the optimum temperature is 30-45℃, and Gram staining is positive.

[0054] The cultivation method of bacillus subtilis comprises the following steps:

[0055] (1) Collect wild-type strains: collect the soil containing Bacillus subtilis, add LB medium according to the ratio of soil to medium amount of 1:100, culture at 37°C, 200rpm for 24 hours, and obtain bacteria rich in Bacillus spores Suspension to obtain the original strain.

[0056] Soil can be collected in virgin forest areas, places rich in degrading bacteria.

[00...

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Abstract

The invention belongs to the field of biotechnology and particularly relates to a method for efficient secretory expression of foreign proteins by use of bacillus. The method is characterized in that target proteins are accumulated intracellularly in quantity and secreted largely into fermentation liquid. The method does not rely on signal peptide or feature structure and is essentially different from existing Sec way, Tat way, Com system and ABC transfer way. When any target proteins are expressed intracellularly in quantity, the target proteins can be secreted extracellularly in quantity through the way, and the maximum secretion quantity can reach a g / L level. The characteristics of small secretion quantity and low universality of existing secretion way are overcome, the target proteins can be secreted into the fermentation liquid non-selectively, and the difficulty of later separation and purification is reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for efficiently secreting and expressing foreign proteins by using bacillus. Background technique [0002] Protein expression technology is one of the core technologies of modern biology. Protein expression can not only be used in biological research, but also provide commercial protein products, such as recombinant vaccines, recombinant insulin, cytokines and other products. Currently commonly used expression systems include Escherichia coli, yeast, insect cells and mammalian cells, etc., but each of them has obvious advantages and disadvantages. Escherichia coli expression system is the most well-studied, and there are many options. The most commonly used is Novagen's pET expression system, which uses bacteriophage T7 RNA polymerase to specifically transcribe the target gene behind the T7 promoter. Under optimal conditions, The target protein can reach more th...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P21/02C12R1/125C12R1/07C12R1/10
CPCC12N15/70C12P21/02
Inventor 任钧唐旭雷蕾樊超柴进凯曹付明范佳曹镜
Owner CHENGDU MYTECH BIOTECH
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