Preparation method for soluble truncated human tumor necrosis factor-related apoptosis inducing ligand (TRAIL) active protein

An active protein, soluble technology, applied in the field of bioengineering, can solve the problems of low soluble protein yield, unsuitable for anti-tumor drugs, high cost, and achieve the effects of high yield, simple procedure and short fermentation time

Inactive Publication Date: 2011-04-20
HUBEI UNIV
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  • Abstract
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Problems solved by technology

When expressed in eukaryotic organisms such as yeast, although a certain amount of TRAIL protein can be obtained, the cost is high and it is easy to cause group modification of TRAIL protein; when expressed in prokaryotic organisms such as Escherichia coli, protein folding...

Method used

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  • Preparation method for soluble truncated human tumor necrosis factor-related apoptosis inducing ligand (TRAIL) active protein
  • Preparation method for soluble truncated human tumor necrosis factor-related apoptosis inducing ligand (TRAIL) active protein
  • Preparation method for soluble truncated human tumor necrosis factor-related apoptosis inducing ligand (TRAIL) active protein

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Embodiment 1

[0029] The amplified recombinant plasmid DNA and pET23a plasmid DNA were double-digested with restriction endonucleases NdeI and XhoI, and the digestion buffer was 10×H reaction buffer. Digest at 37°C for 4 hours. A 533bp DNA fragment encoding the 111-281 amino acid peptide segment of the TRAIL protein and a 3.5kb linear pET23a plasmid DNA were recovered after the digestion mixture was subjected to 1% agarose gel electrophoresis. Then the two recovered DNA fragments were mixed according to the ratio of 5 parts of the target DNA fragment: 1 part of the linear pET23a plasmid DNA, and mixed with T 4 DNA ligase was ligated overnight at 16°C. The ligation reaction mixture was then directly transformed into E.coli BL21 (DE 3 ), positive transformants were screened on LB plates containing 100 μg / ml ampicillin. Express the target protein according to the above step D at 17°C, the soluble target protein in the supernatant is 100%, and 0% in the precipitate. If expressed at 25°C or ...

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Abstract

The invention provides a preparation method for a soluble truncated human tumor necrosis factor-related apoptosis inducing ligand (TRAIL) active protein, comprising the following steps of: (A) designing and synthetizing an oligomerization deoxyribonucleic acid (DNA) single-stranded segment according to the amino acid sequence of the human TRAIL protein issued by a Genebank by the preference of a bacterium genetic code; (B) synthetizing a complete double stranded DNA by three-time polymerase chain reaction (PCR); (C) carrying out amplification by utilizing a T-carrier, and inserting the amplified double stranded DNA segment into an expression carrier and screening a positive transformant; (D) expressing the truncated human TRAIL protein in escherichia coli at low temperature; (E) purifying the protein by using a three-step method; and (F) determining the truncated TRAIL protein. The invention realizes that the truncated human TRAIL protein is efficiently expressed in an escherichia coli cell and a purification preparation technique thereof and overcomes the problem that an infusible inclusion body is easy to form in the prior art. The method has the advantages of simple operation, short fermentation time and easy purification, and moreover, the protein is ensured not to have any modification, and the protein reaches electrophoretically pure. The invention can be directly used for the research work of biochemistry and molecular biology and the oncology and the preclinical stage of tumor treatment or the development of clinical drugs.

Description

technical field [0001] The present invention relates to bioengineering technology, especially the method for expressing and preparing soluble truncated human TRAIL active protein. Background technique [0002] Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also known as apoptin 2 ligand (Apo2ligand, Apo2L). TRAIL is the third member of the tumor necrosis factor superfamily discovered after TNF and FasL. The human TRAIL gene is located on chromosome 3q26, with a full length of 1769bp, encoding 281 amino acids, a protein relative molecular mass of 32.5kd, and an isoelectric point of 7.63, of which 241 amino acids are located outside the cell membrane, called soluble TRAIL fragments, and the functional site is 119-241 amino acids. Both membrane-bound TRAIL and soluble TRAIL protein can induce apoptosis of various tumor cells in vitro, and normal tissues are not sensitive to TRAIL protein. In view of the potential application value of TRAIL protein in tumor ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/70C07K14/705C07K1/36C07K1/30C07K1/20C07K1/18
Inventor 王行国叶青陈凡
Owner HUBEI UNIV
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