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

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 ...

AI Technical Summary

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 is abnormal due to overexpression Formation of inclusion bodies, low yield of soluble protein

Although inclusion bodies can obtain refolded TRAIL protein through denaturation and refolding process, it is only suitable for antibody preparation and not suitable for antitumor drugs

Images