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Producing method for small molecule companion with gene engineering Escherichia coli

A technology of Escherichia coli and genetic engineering, which is applied in the field of genetic engineering Escherichia coli to produce new small molecular chaperones, can solve the problems of limiting GroEL protein renaturation research and industrial application, increasing the difficulty of plasmid construction and soluble expression, and large amount of GroEL, etc. The effect of mild conditions, high level of production automation and short production cycle

Inactive Publication Date: 2003-06-18
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large molecular weight of GroEL, the difficulty of plasmid construction and soluble expression is increased, the extraction process is complicated, and the operating cost is high. However, the amount of GroEL used in renaturation is relatively large (the molar ratio to the target protein is generally ≥ 1), which severely limits Research and industrial application of GroEL in vitro assisted protein renaturation

Method used

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  • Producing method for small molecule companion with gene engineering Escherichia coli
  • Producing method for small molecule companion with gene engineering Escherichia coli
  • Producing method for small molecule companion with gene engineering Escherichia coli

Examples

Experimental program
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Effect test

Embodiment 1

[0054] The steps of the method for producing novel small molecule chaperones with genetically engineered Escherichia coli are as follows:

[0055] 1) Engineering bacteria fermentation

[0056] Pick a single bacterium colony from the solid medium and insert it into 50mL seed medium containing ampicillin, so that the bacterium concentration in the medium reaches 100mg / L, cultivate it in a 180rpm shaker at 33°C for 12 hours, and inoculum by 2% The previous culture solution was inserted into the seed medium containing ampicillin again, and cultivated for 10 hours at 33° C. in a shaker at 180 rpm, then the activated seed solution was added to the fermentation medium by 2% inoculum size, and ampicillin was added simultaneously to make The final concentration was 50 μg / mL, cultured in a shaker at 33°C and 180 rpm for 3 hours, then added placebo inducer isopropyl-β-D-thiogalactoside with a final concentration of 0.5 mM for induction, and continued to culture for 8 Hours, the new smal...

Embodiment 2

[0066] The steps of the method for producing novel small molecule chaperones with genetically engineered Escherichia coli are as follows:

[0067] 1) Engineering bacteria fermentation

[0068] Pick a single colony from the solid medium with a toothpick and insert it into 10mL of ampicillin-containing seed medium, so that the bacterial concentration in the medium reaches 100mg / L, culture in a shaker at 33°C and 180rpm for 16 hours, and inoculate at 1%. Add the previous culture solution to the seed medium containing ampicillin again, cultivate it in a shaker at 33°C and 180rpm for 12 hours, then add the activated seed solution to the fermentation medium according to the inoculum size of 1%, and add ampicillin at the same time Penicillin made the final concentration of 50μg / mL, cultured in a shaker at 33°C and 180rpm for 4 hours, then added placebo inducer isopropyl-β-D-thiogalactoside with a final concentration of 0.2mM for induction, continued After culturing for 7 hours, the ...

Embodiment 3

[0078] The steps of the method for producing novel small molecule chaperones with genetically engineered Escherichia coli are as follows:

[0079] 1) Engineering bacteria fermentation

[0080] Pick a single bacterium colony from the solid medium and insert it into 100mL seed medium containing ampicillin, so that the bacterium concentration in the medium reaches 200mg / L, cultivate it in a shaker at 250rpm at 37°C for 24 hours, and inoculum by 0.5% The previous culture solution was inserted into the seed culture medium containing ampicillin again, and cultivated in a shaker at 250 rpm for 12 hours at 37° C., then the activated seed solution was added to the fermentation medium by 0.5% inoculum size, and ampicillin was added simultaneously to make The concentration was 50 μg / mL, cultured in a shaker at 37°C and 250 rpm for 6 hours, then added the placebo inducer isopropyl-β-D-thiogalactoside with a final concentration of 1 mM for induction, and continued to culture for 9 hours. ...

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Abstract

A process for preparing novel micro-molecular mate by use of genetically engineered colibacillus includes such steps as activating the genetic engineering bacterial seeds in culture medium, culturing in fermented culture medium to accumulate the micro-molecular mate in the bacterial cells, centrifugal separating of bacterial cells, resuspending with buffer liquid, ultrasonic breaking of cells to release the micro-molecular mate into solution, centrifugal separating, collecting supernatant, affinity adsorbing by affinity chromatograph column, eluting, gel separating by gel column, and freeze drying at -40 deg.C. Its advantages are high effect, low cost and no environmental pollution.

Description

technical field [0001] The invention relates to a method for producing novel small molecular chaperones by using genetically engineered Escherichia coli. Background technique [0002] Molecular chaperones are a class of proteins that are not related to each other, and their function is to help substances with polypeptide structures to carry out correct non-covalent assembly in vivo. This name was first proposed by Laskey in 1978, but it was not until 1987 that Ellis formally gave the concept of molecular chaperones that molecular chaperones assist in the folding and assembly of proteins were truly recognized and valued. Molecular chaperones belong to heat shock proteins (Heat Shock Proteins, Hsp), mainly including three protein families of chaperone proteins Hsp60, Hsp70 and Hsp90. The interior of the cell is a highly crowded environment for macromolecules, and the protein concentration can reach about 300mg / mL. Such a high concentration increases th...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P21/02
Inventor 关怡新姚善泾高永贵张佳艺
Owner ZHEJIANG UNIV
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