Expression, Purification Of Recombinant Human Tumor Necrosis Factor-related Apoptosis Ligand (rhTRAIL) Protein And Its Activity

Tumor necrosis factor-related apopsis ligand(TRAIL), also known as Apo2 ligand, is a new member of the TNF superfamily. Its soluble form can induce apoptosis of various cancer cell lines while has minimal cytotoxicity toward normal tissues both in vitro and in vivo. Human TRAIL precursor is a 281 aa type II transmembrane protein, which contains a short N-terminal intracellular domain, a transmembrane domain and a C-terminal extracellular domain. Its molecular weight is 32.5kD and PI is 7.63. Three soluble molecules could bind a Zn²⁺ to form homotrimer.TRAIL-induced apoptosis is mediated by its receptors. To date, five TRAIL receptors have been identified: TRAIL-R1 (DR4), TRAIL-R2 (DR5), TRAIL-R3 (DcR1), TRAIL-R4 (DcR2) and osteoprotegerin (OPG). Engagement of TRAIL-R1 or TRAIL-R2 by TRAIL results in activation the death receptor pathway and the mitochondrial pathway. Both the death receptor and the mitochondrial pathway activatied caspase-3 and result in apoptosis. TRAIL has a great deal of attention as a potential anti-tumor agent.In our study, we investigated expression, purification of rhTRAIL_114-281 prote and its inhibition on tumor cells. First of all, We choiecd E.coli w3110 as host and transformed the pET-23d(+)/TRAIL_114-281 into w3110.Subsequently, in order to increased the expression level of TRAIL, we investigated different fementation conditions in shake flask. The target protein was then identified by SDS-PAGE and analysed. rhTRAIL_114-281 protein expression yield was up to 50% in the total proteins, and soluble form of rhTRAIL_114-281 protein was up to 20% of supernatant protein. The total protein was purified by Q Sepharose XL and Sourcel5Q anion-exchange chromatography. The purity of protein TRAIL reached to 96%, detected by HPLC. At last, Antitumor activity of rhTRAIL protein was evaluated by MTT method. Results showed that purified rhTRAIL_114-281 protein could inhibited the proliferation of KB_v200, A549, K562 and SMMC 7721 tumor cells.