Cloning, Expression Of TRAIL CDNA And Purification Of The Protein And Its Biological Function Research

TRATJL(TNF-related apoptosis-inducing lligand) was discovered because of its sequence homology to TNF and FasL, which belongs to type II transmembrane protein as other members of TNF family. Human TRAIL contains 281 aa, of which 14 aa in N-termini consists of its cytoplasm region, following 26 aa makes its transmembrane region and a 241 aa extracellular region. Its molecular weight is 32.5 kD, IE is 7.63. The extracellular region of TRAIL forms a soluble molecule upon cleavage. This molecule usually forms stable homotrimers or homodimers. In contrast to TNF and FasL, TRAIL mRNA is expressed constitutively in many tissues, which suggests the existence of physiological mechanism that can protect many normal cell types from induction of apoptosis specially by TRAIL.TRAIL induces apoptosis through the interaction of TRAIL and its receptors So far, we have found five receptors of TRAIL : DR4(TRAIL-R1)N DR5(TRAIL-R2)^ DcRl(TRAIL-R3) , DcR2(TRATL-R4) and OPG. DR4 and DR5 have completely cytoplasm death domain and can induce apoptosis when TRAIL bind to them. But DcRl > DcR2 and OPG can not induce apoptosis because they have not death domain in cytoplasm and can not transform the signal of apoptosis. DR4 and DR5 are expressed broadly in many tissues, such as spleen liver > lung^ thymus ^ PBC > activated T cells and some tumor cells. DcRl is expressed only in normal tissue, not in tumor tissues, while DcR2 is expressed only in embryonic liver tissue and adult testis tissue. The binding of8 Biotechnology Center FMMUTRAIL to DR4 and DR5 can induce apoptosis while to DcRl and DcR2 can protect the cells from apoptosis. Because many tumor cells only express DR4 and DR5 and don't express DcRl and DcR2, TRAIL can specifically kill tumor cells while have no toxicity to normal tissues. It was confirmed that TRAIT, can kill various tumor cells such as leukocyte cancer cells > breast cancer cells and so on So TRAIL is considered as a potential drug in tumor therapy.To further investigate the mechanism of how TRAIL inducing apoptosis and the prospect of TRAIL as a clinic drug in cancer therapy, we amplified 510 bp cDNA fragment of the solible region of TRAIL from PBC by RT-PCR. After DNA sequencing in pGEM3zf/sTRAIL, sTRAIL cDNA was cloned into expression vector pBV220, then transformed into E.coli DH5 a . TRAIT, was highly expressed after 4 hours' induction at 39 癈. SDS-PAGE indicated that the quantity of TRAIL protein is 25.6% of total bacterial protein and part of the TRAILis in soluble form. To purify the soluble TRAIL from E.coli, The supernatant of the bacterial lysate was separated sequencially by SP-Sepharose FF positive ion chromatography and Q-Sepharose FF negative ion chromatography. We obtained TRAIL protein with high purification (94.3%) and confirmed its bioactivity with L929 cell line in vitro. To investigate the bioactivity of TRAIL, we established animal tumor models in KM mice by inoculating SI80 cells and applied TRAIL to them. The result indicated that lOmg/kg TRAIL can inhibit tumor growth by 67.2% and was better than the positive control(52.2%).