Expression And Biological Function Of Recombinant Human TRAIL And Its Mutants

TRAIL/Apo2 (TNF-related apoptosis inducing ligand), a novel cytokine, is a member of the tumor necrosis factor(TNF) family and was first cloned and identified by Willy in 1995, which can be detected in a varity of human tissue, most predominantly in spleen, lung, prostate,thymus and peripheral blood lymphocytes. TRAIL consists of 281 amino acids, and its gene is located on chromosome 3 at position 3q25. TRAIL as well as other members of TNF family, including TNF α , lymphotoxin β (LT β ),and ligands for CD40, CD30, CD27,OX40L is type II membrane protein, which can be hydrolyzed to soluble homotrimers. TRAIL play an important role in regulating many biological functions, such as prominent mediators of immune regulation and inflammatory responses, especially in inducing apoptosis in a wide variety of transformed cell lines and tumor cells. The key point between TRAIL and other apoptosis inducing ligands of the TNF family is that TRAIL effectively kills many tumor cells by apoptosis while leaving normal cells unharmed. These cytotoxic lignads induce the apoptosis (programmed cell death) by binding the receptors, which contain death domains.The full-length of TRAIL oligonucleotide was synthesized, and prokaryotic expression vector was constructed. After gene transfecting, the stable expression E coli DH-5α were obtained. Fusion TRAIL protein was expressed in E coli DH-5 α and purified by affinity chromatography. Subsequently, the purified protein wastested to assess its ability to induce apoptosis in various tumor cell lines such as BGC832 lung cancer and A172 glioma cells. We also got two TRAIL mutants through homology modeling. These two TRAIL mutants were expressed in E coli DH-5 α ,after their oligonucleotide, fragments were inserted into pBV220, a thermo-sensitive expression vector.The synthesis of full-length of TRAIL oligonucleotides, expression of TRAIL in E coli DH-5α and purification of TRAIL: The full-length of human TRAIL oligonucleotide codes for 841 bases, which was synthesized and thirty of which were replaced to make TRAIL expressed in E coli DH-5α efficiently. The DNA sequence analysis proved the full-length of human TRAIL oligonucleotide synthesized was the same as which we wanted. The mutant TRAIL oligonucleotide was inserted into pGEX-2T vector, which contains a GST protein code to be purified easily by affinity chromatography, then transfecting E coli DH-5 a . The fusion TRAIL protein was expressed in E coli DH-5 a induced by 0.4mM/L IPTG for 4 hours at 28℃, and purified by affinity chromatography through GSTrap column.Antitumor activity of TRAIL: The cytotoxic activities of TRAIL were measured on A172 huanm glioma cells, B104 murine neuroblastoma, SKNSH human neuroblastoma, BGC823 stomach cancer cells, A549 lung cancer cells, H69 lung cancer cells, KB nasopharyngeal cancer cells, etc. TRAIL to a final concentration of 1μg/ml is capable to induce apoptosis of these tumor cell lines efficiently.TRAIL mutant design based on homology modeling: TRAIL belongs to a member of TNF family, which gene codes for a polypeptide of 281 amino acids. The soluble active extracellular segment of TRAIL ranged from amino acids 95-281. The two TRAIL mutants were obtainedby homology modeling, one is residues 130-145 and 210-281, and the other is residues 145-281. The primers were designed according to oligonuclotide sequence of TRAIL mutants, 5' primer with EcoR I restriction enzyme site and 3' primer with BamH I restriction site. The mutants oligonucleotide segments were obtained by PCR, and then inserted into pUC18-T vector. The result of sequence analysis of mutants proved properly.Expression and purification of TRAIL mutants: The mutants oligonucleotide segments were inserted into pBV220.The recombinant plasmid pBV-TRAIL mutant identified by digestion with EcoR I AND BamH I was transformed to the competent cells of E coli DH-5α , and then the transformed cells were induced to be expressed by shifting culture from 37℃ to 42℃. The TRAIL mutants were expressed in the form of inclusion bodies and soluble protein. The inclusion bodies were washed with 8M urea, and then purified preparatively.