Expression of soluble TRAIL protein

A soluble protein technology, applied in the field of bioactive protein expression, can solve the problems of high inclusion body content and low soluble TRAIL protein content, and achieve the effect of increasing expression and reducing the ratio

Inactive Publication Date: 2009-06-17
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a method for expressing TRAIL protein using Escherichia coli to overcome the problems of high content of inclusion bodies and low content of soluble TRAIL protein in the prior art

Method used

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  • Expression of soluble TRAIL protein
  • Expression of soluble TRAIL protein
  • Expression of soluble TRAIL protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0052] Example 2. Using 38°C as the induction temperature to induce the expression of TRAIL protein

[0053] After the recombinant Escherichia coli strain C600-TRAIL was activated in 30mL LB medium at 30°C, it was inoculated into 30mL LB medium at 1% inoculum size and cultured at 30°C. Before inoculation, ampicillin should be added to a concentration of 100μg / mL. Grow to OD 600 0.60, the shaker flask was placed in a shaker at 38°C for 8 hours, and the rotation speed was 200rpm during the growth phase and the induction phase.

[0054] Among them, the composition of LB medium is 10g / L peptone, 5g / L yeast powder, 5g / L sodium chloride, and the pH is adjusted to 7.0.

[0055] Treat the thalli in the same manner as in Example 1, carry out SDS-PAGE electrophoresis to the supernatant and the whole bacterium lysate, and scan the electrophoretic pattern to quantify the target product, see figure 1 . Compared with the usual induction expression at 42°C, when 38°C is used as the induct...

Embodiment 3、38

[0056] Example 3, 38 degree induction, adding 1mmol / L zinc ion improves the soluble expression of recombinant TRAIL protein

[0057] After the recombinant Escherichia coli strain C600-TRAIL was activated in 30mL LB medium at 30°C, it was inoculated into 30mL LB medium at an inoculum size of 1%, and cultured at 30°C. Before inoculation, ampicillin was required to be added to a concentration of 100 μg / mL. Grow to OD 600 0.585, divalent zinc ions were added under aseptic conditions to a concentration of 1 mmol / L, and the shake flask was placed in a shaker at 38°C for induction. The speed of the growth phase and the induction phase were both 200 rpm, and the induction was performed for 8 hours.

[0058] Among them, the composition of LB medium is 10g / L peptone, 5g / L yeast powder, 5g / L sodium chloride, and the pH is adjusted to 7.0. Using ZnSO 4 aqueous solution.

[0059] Treat the thalli in the same manner as in Example 1, carry out SDS-PAGE electrophoresis to the supernatant ...

Embodiment 5、38

[0064] Example 5, 38-degree induction, adding tetracycline that inhibits protein synthesis improves the soluble expression of recombinant TRAIL protein

[0065] After the recombinant Escherichia coli strain C600-TRAIL was activated in 30mL LB medium at 30°C, it was inoculated into two parts of 30mL LB medium according to the inoculum size of 1%, and cultured at 30°C. Before inoculation, it was necessary to add ampicillin to The concentration is 100 μg / mL. Two cultures were grown separately to OD 600 They were 0.530 and 0.562, respectively, and tetracycline was added under sterile conditions to a concentration of 4 μg / mL and 8 μg / mL. The shake flask was placed in a shaker at 38° C. for induction, and the rotation speed of both the growth phase and the induction phase was 200 rpm, and the induction was performed for 8 hours.

[0066] Among them, the composition of LB medium is 10g / L peptone, 5g / L yeast powder, 5g / L sodium chloride, and the pH is adjusted to 7.0. Antibiotics a...

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Abstract

The invention opened a method to express the soluble TRAIL protein which includes cloning the extracellular gene of the TRAIL into the expression carrier with the PL promotor of the lambda bacteriophage; Transforming the cI+ host bacteria; expressing the recombinative TRAIL protein by the low-temperature induction. Also it can add the Zn2+ or the synthetic inhibiter of the prokaryotic protein into the medium in the inducing stage. The recombinative TRAIL protein is soluble, so it can simplify the isolation process.

Description

technical field [0001] The invention relates to a method for expressing biologically active proteins, in particular to a method for expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein. technical background [0002] Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the newly discovered TNF family members, also known as apoptotin 2ligand (Apo-2L). In 1996, Pitti first isolated and identified TRAIL and its induced apoptosis, and found that the full-length human TRAIL gene was 1769bp, located on chromosome 3q26. TRAIL protein is a type II transmembrane protein, consisting of 281 amino acids, with a molecular weight of 32.5kD and an isoelectric point of 7.63. functional parts. The biologically active form of TRAIL is a homotrimer, and there is a zinc ion binding site on the monomer interface (Sarah G et al., Biochem, 2000, 39:633-640). [0003] The combination of the extracellular region of TRAIL and its specific receptor ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N15/12C12N15/63C12N15/70C12N1/21C12P21/02C07K14/435C12R1/19
Inventor 沈亚领魏东芝孙爱友汤亚南周文瑜
Owner EAST CHINA UNIV OF SCI & TECH
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