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

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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 amou

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
Comparison scheme
Effect test

Example Embodiment

[0053] Example 1

[0054] The steps of using genetically engineered Escherichia coli to produce new small molecule chaperones are as follows:

[0055] 1) Fermentation of engineered bacteria

[0056] Pick a single colony from the solid medium and put it into 50mL seed medium containing ampicillin, make the cell concentration in the medium reach 100mg / L, culture in a shaker at 33°C and 180rpm for 12 hours, and add 2% inoculum to The previous culture liquid was again connected to the seed culture medium containing ampicillin, cultured in a shaker at 33°C and 180rpm for 10 hours, and then the activated seed liquid was added to the fermentation medium according to the 2% inoculum, and ampicillin was added at the same time to make The final concentration is 50μg / mL, 33℃, 180rpm shaker for 3 hours, then add the placebo inducer isopropyl-β-D-thiogalactoside at a final concentration of 0.5mM for induction, continue to culture 8 When hours, new small molecular chaperones accumulate in the b...

Example Embodiment

[0065] Example 2

[0066] The steps of using genetically engineered Escherichia coli to produce new small molecule chaperones are as follows:

[0067] 1) Fermentation of engineered bacteria

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

Example Embodiment

[0077] Example 3

[0078] The steps of using genetically engineered Escherichia coli to produce new small molecule chaperones are as follows:

[0079] 1) Fermentation of engineered bacteria

[0080] Pick a single colony from the solid medium and insert it into 100mL seed culture medium containing ampicillin, so that the bacterial cell concentration in the medium reaches 200mg / L, 37℃, 250rpm shaker for 24 hours, and 0.5% inoculum The previous culture solution was again connected to the seed culture medium containing ampicillin, cultivated in a shaker at 37°C and 250 rpm for 12 hours, and then the activated seed solution was added to the fermentation medium according to the 0.5% inoculum, and ampicillin was added at the same time to make The concentration is 50μg / mL, 37°C, 250rpm shaker for 6 hours, then add the placebo inducer isopropyl-β-D-thiogalactoside with a final concentration of 1mM to induce, continue to cultivate for 9 hours, New small molecular chaperones accumulate in bact...

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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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