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Method for enhancing stability of escherichia coli T7 expression system on basis of double-repression strategy

An Escherichia coli, expression system technology, applied in the field of Escherichia coli expression regulation, can solve the problems of weakened expression, low expression of target protein, and slow growth of strains

Active Publication Date: 2014-02-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, although the background expression level of the BL21(DE3) pLysS strain is almost only one-tenth of that of the BL21(DE3) strain, the background expression of the former still exists and may still cause system instability
Moreover, T7 lysozyme is a bifunctional protein that has a damaging effect on the cell wall of E. coli, so strains containing pLysS or pLysE plasmids grow slower
In addition, after induction, the expression of T7 lysozyme will be weakened, so that the expression level of the target protein is very low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0, prepared with double distilled water. When needed, add ampicillin (100 μg / mL) or kanamycin (50 μg / mL) before use, and add 15 g / L agar powder to the solid medium.

[0057] Pick newly converted E. coli BL21(DE3) / pET-20b(+)- pul A single colony was inoculated in 3 mL of LB liquid medium containing kanamycin (50 μg / mL), and cultured overnight at 37 °C with shaking at 200 rpm. Take 0.5 mL of the culture medium and transfer it to 50 mL of LB liquid medium containing the corresponding antibiotics, shake and culture at 37°C and 200 rpm until OD 600 About 1.2. The culture solution was transferred to 20° C. to continue culturing for 12 hours. After the cultivation, the fermentation broth was centrifuged at 10,000 rpm for 10 minutes, the cell pellet was ultrasonically broken, and the crushed solution was centrifuged at 12,000 rpm for 20 minutes, and the supernatant was taken as the intracellular component,...

Embodiment 2

[0059] LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0, prepared with double distilled water. When needed, add ampicillin (100 μg / mL) or kanamycin (50 μg / mL) before use, and add 15 g / L agar powder to the solid medium.

[0060] Recombinant plasmid pET-20b(+)- pul convert E. coli BL21 competent cells were inoculated in 3 mL of LB liquid medium containing corresponding antibiotics immediately after the transformants grew out, and cultured overnight at 37°C with shaking at 200 rpm. Take 0.5 mL of the culture medium and transfer it to 50 mL of LB liquid medium containing the corresponding antibiotics, shake and culture at 37°C and 200 rpm until OD 600 About 1.2. The culture solution was transferred to 20° C. to continue culturing for 12 hours. After the cultivation, the fermentation broth was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was retained as the extracellular component. The cell pellet was ultrasonically disrupted, the disrup...

Embodiment 3

[0062] Autoinduction medium (g / L): β-lactose 1~50, anhydrous glucose 0.5, glycerol 5, KH 2 PO 4 6.8, MgSO 4 0.24, tryptone 10, yeast extract 5, Na 2 HPO 4 7.1, Na 2 SO 4 0.71, NH 4 Cl 2.67, trace element solution 400 μL / L, pH 7.5~8.0, prepared with double distilled water.

[0063] Trace element solution (g / L): FeCl 3 8.125, CaCl 2 2.22, MnCl 2 2.52, ZnSO 4 1.61, CoCl 2 0.26, CuCl 2 0.27, NiCl 2 0.26, Na 2 MoO 4 0.41, Na 2 SeO 3 0.346,H 3 BO 3 0.124, HCl 2.19, prepared by double distilled water.

[0064] Recombinant plasmid pET-22b(+)- pul convert E. coli BL21 competent cells were inoculated in 3 mL of LB liquid medium containing corresponding antibiotics immediately after the transformants grew out, and cultured overnight at 37°C with shaking at 200 rpm. Take 2 mL of the culture solution and inoculate it into 50 mL of the autoinduction medium containing the corresponding antibiotics, culture at 37 °C and 200 rpm for 2 hours, then transfer...

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Abstract

The invention relates to a method for enhancing the stability of an escherichia coli T7 expression system on the basis of a double-repression strategy, and belongs to the field of regulation and control of escherichia coli expression. The method disclosed by the invention can be used for constructing the recombinant escherichia coli which expresses pullulanase by utilizing a gene engineering technology and taking the pullulanase of a bacteria source as an expression protein and constructing the recombinant bacteria E.coliBL21 / pET-22b(+)-pul and E.coliBL21 / pET-28a(+)-PelB-pul by using PET expression vectors pET-22b(+) and pET-28a(+) which contain lac operons and repressor genes lacI, basically eliminates the background expression of a toxic foreign protein by utilizing the precise double-repression regulation and control strategy of the lac operons, enhances the expression stability of a frozen glycerol strain and the expression quantity of target proteins with lactose self-induced and enhances the stability of the escherichia coli T7 expression system. According to the invention, the common problem of system stability of the escherichia coli T7 expression system is solved, and the method for enhancing the stability of the escherichia coli T7 expression system is developed.

Description

technical field [0001] The invention relates to a method for enhancing the stability of an Escherichia coli T7 expression system based on a double-repression strategy, which belongs to the field of expression regulation of Escherichia coli. Background technique [0002] Recombinant technology has been widely used for high-level expression of target proteins. Among the various systems available for expression of heterologous proteins, the E. coli system remains the most commonly used expression host. Compared with other expression hosts, Escherichia coli has many advantages, such as clear genetic background, rapid growth, high density in inexpensive media, and the ability to overexpress target proteins. The T7 system based on T7 RNA polymerase can express the target protein amount up to 50% of the total cell protein amount, thus showing advantages over other E. coli systems. [0003] The speed of T7 RNA polymerase to synthesize mRNA is 5 times that of Escheric...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12R1/19
Inventor 聂尧徐岩
Owner JIANGNAN UNIV
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