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Method for producing neoplasm metastasis inhibiting protein GDI2 through escherichia coli accumulation

A tumor metastasis, Escherichia coli technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as the decline of fermentation yield, and achieve the effect of high yield and reduced accumulation

Active Publication Date: 2013-08-14
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, in the fermentation model of recombinant protein produced by Escherichia coli, the fed-batch fed-batch strategy has become a common strategy to increase the concentration of bacteria and protein products, but in the fermentation process of Escherichia coli using this strategy, it cannot fundamentally Addresses issues such as metabolic by-product accumulation (acetic acid), which ultimately leads to a decrease in fermentation yield

Method used

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  • Method for producing neoplasm metastasis inhibiting protein GDI2 through escherichia coli accumulation

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Experimental program
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Effect test

Embodiment 1

[0024] The method of accumulatively producing tumor metastasis suppressor protein GDI2 using E. coli includes the following steps:

[0025] A single clone of E. coli engineered bacteria was selected and cultured in LB medium (containing 50 μg / ml of ampicillin) at 37° C. and a rotation speed of 200 rpm / min for 12-14 hours as the first-level seeds. Then, inoculate the secondary seed vial medium (g / L) according to the 3% inoculum: peptone 10g / L, yeast powder 5g / L, glucose (C 6 H 12 O 6 ·H 2 O) 2g / L, NaCl 1g / L, NaH 2 PO 4 ·12H 2 O 2g / L, Na 2 HPO 4 ·3H 2 O 7g / L, K 2 HPO 4 ·3H 2 O 4g / L, ZnSO 4 ·7H 2 O 0.1g / L, MgSO 4 ·7H 2 O 1g / L, pH 7.0. Cultivate for 8 hours at 37°C and rotation speed 200r / min, and inoculate it into a 3.7L fermentor at an inoculum amount of 3% based on the volume of the medium.

[0026] Optimized fermentation medium (g / L) was used in the initial stage of fermentation: peptone 10g / L, yeast powder 10g / L, glucose (C 6 H 12 O 6 ·H 2 O) 2g / L, NaCl 1g / L, NaH 2 PO 4 ·12H 2 O 2g...

Embodiment 2

[0030] The method of accumulatively producing tumor metastasis suppressor protein GDI2 using E. coli includes the following steps:

[0031] A single clone of E. coli engineered bacteria was selected and cultured in LB medium (containing 50 μg / ml of ampicillin) at 37° C. and a rotation speed of 200 rpm / min for 12-14 hours as the first-level seeds. Then, inoculate the secondary seed vial medium (g / L) according to the 3% inoculum: peptone 10g / L, yeast powder 5g / L, glucose (C 6 H 12 O 6 ·H 2 O) 2g / L, NaCl 1g / L, NaH 2 PO 4 ·12H 2 O 2g / L, Na 2 HPO 4 ·3H 2 O 7g / L, K 2 HPO 4 ·3H 2 O 4g / L, ZnSO 4 ·7H 2 O 0.1g / L, MgSO 4 ·7H 2 O 1g / L, pH 7.0. Cultivate for 8 hours at 37°C and rotation speed 200r / min, and inoculate it into a 3.7L fermentor at an inoculum amount of 3% based on the volume of the medium.

[0032] Optimized fermentation medium (g / L) is used in the initial stage of fermentation: peptone 15g / L, yeast powder 10g / L, glucose (C 6 H 12 O 6 ·H 2 O) 4g / L, NaCl 1g / L, NaH 2 PO 4 ·12H 2 O 2g / ...

Embodiment 3

[0036] The method of accumulatively producing tumor metastasis suppressor protein GDI2 using E. coli includes the following steps:

[0037] A single clone of E. coli engineered bacteria was selected and cultured in LB medium (containing 50 μg / ml of ampicillin) at 37° C. and a rotation speed of 200 rpm / min for 12-14 hours as the first-level seeds. Then, inoculate the secondary seed vial medium (g / L) according to the 3% inoculum: peptone 10g / L, yeast powder 5g / L, glucose (C 6 H 12 O 6 ·H 2 O) 2g / L, NaCl 1g / L, NaH 2 PO 4 ·12H 2 O 2g / L, Na 2 HPO 4 ·3H 2 O 7g / L, K 2 HPO 4 ·3H 2 O 4g / L, ZnSO 4 ·7H 2 O 0.1g / L, MgSO 4 ·7H 2 O 1g / L, pH 7.0. Cultivate for 8 hours at 37°C and rotation speed 200r / min, and inoculate it into a 3.7L fermentor at an inoculum amount of 3% based on the volume of the medium.

[0038] Optimized fermentation medium (g / L) is used in the initial stage of fermentation: peptone 15g / L, yeast powder 10g / L, glucose (C 6 H 12 O 6 ·H 2 O) 2g / L, NaCl 1g / L, NaH 2 PO 4 ·12H 2 O 2g / ...

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Abstract

The invention relates to a method for producing neoplasm metastasis inhibiting protein GDI2 through escherichia coli accumulation, which comprises the steps that: after primary and secondary seed culture is carried out on constructed escherichia coli, secondary seed culture liquid is inoculated into a fermentation tank according to the inoculation quantity of 3wt percent, the fermentation temperature is controlled to be 37 DEG C, the pH is 7.0, supplemented culture media are started to be added when the dissolved oxygen in fermentation liquid is obviously increased so that thalli grow under the condition of the constant specific growth rate, the fermentation with high cell density is finally realized, the final germ concentration OD can reach 116, and the neoplasm metastasis inhibiting protein GDI2 is obtained after 0.2 mM IPTG inducement for 4h. Compared with the prior art, the method has the advantages that the non-most economic section in the fermentation process can be effectivelyreduced, in addition, the fermentation strategy control is simple, the target protein yield is very high, and higher industrial application prospects are realized.

Description

Technical field [0001] The present invention relates to a fermentation production method of tumor metastasis suppressor protein GDI2, in particular to a method for accumulatively producing tumor metastasis suppressor protein GDI2 using Escherichia coli. Background technique [0002] Rho-GDP dissociation inhibitor 2 (GDI2), first discovered by Leffers et al., is composed of 201 amino acids and is a member of the Rho GDP dissociation inhibitor (RhoGDI) family. In recent years, a large number of studies have proved that the RhoGDI2 protein is responsible for preventing the spread of cancer cells throughout the body in the human body. The protein can effectively inhibit the invasion and metastasis of tumor cells, and endothelin 1 (ET-1) and neural mediators Element U (NmU) may be its target. After GDI2 is cleaved by caspase-1 in human body, the 21kDa GDI2 short peptide can greatly increase the apoptosis of cancer cells through the anoikis mechanism (Anoikis), thereby inhibiting cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C12R1/19
Inventor 孙爱友魏东芝王学东徐瑞董玉国张星王丽华
Owner EAST CHINA UNIV OF SCI & TECH