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Recombinant secretory escherichia coli high-density fermentation method

A high-density fermentation and Escherichia coli technology, applied in the field of microbial fermentation, can solve the problems of high requirements for operators and equipment, complex preservation methods, and high costs, and achieve the effects of shortening the fermentation cycle, increasing bacterial growth density, and improving stability.

Inactive Publication Date: 2017-12-26
德清知诺同丰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method adopts a three-level strain preservation method, which increases the effective storage period to a certain extent and reduces the probability of abnormal production due to strain degradation during the fermentation process, but the probability of expressing the target protein by the strain used still depends on the construction. coli, and the preservation method is complex, involving processes such as subpackaging under aseptic conditions and vacuum freeze-drying, which have high requirements for operators and equipment, high costs, and increase the risk of damage caused by the entry of foreign bacteria. Risk of Abnormal Species

Method used

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  • Recombinant secretory escherichia coli high-density fermentation method

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Effect test

Embodiment 1

[0025] The method for high-density fermentation of recombinant secretory Escherichia coli comprises the following steps:

[0026] (1) The constructed recombinant secretory Escherichia coli was preserved as the original strain. Inoculate the constructed recombinant secretory Escherichia coli at a ratio of 1% in 50ml LB liquid medium containing antibiotics, culture it on a constant temperature air shaker at 37°C and a speed of 200 rpm for 8 hours, and measure the OD 600 The value was 2.5. After sampling and staining microscopic examination to confirm that there was no bacterial contamination, the bacterial solution was mixed with 30% glycerol at a ratio of 1:1, sealed with a parafilm, and stored in a -80°C refrigerator.

[0027](2) Screen high-expression strains. The constructed recombinant secretory Escherichia coli was inoculated at 1:10 in 50ml liquid screening medium containing antibiotics; the liquid screening medium included: yeast powder 0.06%, peptone 0.12%, sodium chlo...

Embodiment 2

[0035] The method for high-density fermentation of recombinant secretory Escherichia coli comprises the following steps:

[0036] (1) The constructed recombinant secretory Escherichia coli was preserved as the original strain. Inoculate the constructed recombinant secretory Escherichia coli at a ratio of 1% in 50ml LB liquid medium containing antibiotics, culture it on a constant temperature air shaker at 37°C and a speed of 200 rpm for 4 hours, and measure the OD 600 The value is 2.0. After sampling and staining microscopic examination to confirm that there is no contamination by bacteria, take the bacterial solution and 30% glycerol and mix them at a ratio of 1:1, then seal them with a parafilm, and store them in a -80°C refrigerator.

[0037] (2) Screen high-expression strains. The constructed recombinant secretory Escherichia coli was inoculated at 1:10 in 50ml liquid screening medium containing antibiotics; the liquid screening medium included: yeast powder 0.04%, pepton...

Embodiment 3

[0043] The method for high-density fermentation of recombinant secretory Escherichia coli comprises the following steps:

[0044] (1) The constructed recombinant secretory Escherichia coli was preserved as the original strain. Inoculate the constructed recombinant secretory Escherichia coli at a ratio of 1% in 50ml LB liquid medium containing antibiotics, culture it on a constant temperature air shaker at 37°C and a speed of 200 rpm for 12 hours, and measure the OD 600 The value is 3.0. After sampling and staining microscopic examination to confirm that there is no contamination by bacteria, take the bacterial solution and 30% glycerol in a ratio of 1:1 and mix it with a parafilm, then store it in a -80°C refrigerator.

[0045] (2) Screen high-expression strains. The constructed recombinant secretory Escherichia coli was inoculated in 50ml liquid screening medium containing antibiotics at a ratio of 1:10; the liquid screening medium included: yeast powder 0.08%, peptone 0.14%...

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Abstract

The invention discloses a recombinant secretory escherichia coli high-density fermentation method. The method comprises the steps of saving original strain of constructed recombinant secretory escherichia coli; screening high-expression strain; conducting working strain saving on the screened high-expression strain; activating the working strain; transplanting the working strain into a shake flask to expand cultivation; inoculating a fermentation tank to carry out high-density fermentation; controlling oxygen dissolve conditions by the adjustment of tank pressure, ventilation volume, stirring speed and material supply speed; according to the changes of oxygen dissolve, a pH value and the like, adjusting fed-batch speed of a supplemented medium; after the strain grows to high density, adjusting strain growing speed by controlling the fed-batch speed of the supplemented medium, and cooling to carry out fermentation until the fermentation is finished. The recombinant secretory escherichia coli high-density fermentation method has the advantages of being high in objective protein expression probability, high in growth density, short in fermentation period, easy to operate, low in cost and significantly reducing the risks of abnormal strain caused by external strain entering.

Description

technical field [0001] The invention relates to the field of microbial fermentation, in particular to a method for high-density fermentation of recombinant secretory Escherichia coli. Background technique [0002] Protein secretory expression means that recombinant heterologous protein is localized in the periplasm of the cell through transport or secretion, and even enters the medium through the outer membrane. The advantage of secretory expression of recombinant protein is that in the process of periplasmic or extracellular secretory expression, the signal peptide is cleaved in the cell, which is more likely to produce the natural N-terminal of the target protein. If it contains methionine, it may reduce the solubility and stability of the product. Stability, the periplasm of Escherichia coli contains a series of enzymes, and provides an oxidative environment, which is conducive to the correct formation of disulfide bonds, and enhances the correct folding of sulfhydryl pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N1/04C12R1/19
CPCC12N1/20C12N1/04
Inventor 雷振松王勇波
Owner 德清知诺同丰生物科技有限公司
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