| Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the several members of the necrosis factor (TNF) gene superfamily that induce apoptosis through engagement of death receptors (DRs).TRAIL interacts with an unusually complex receptor system of two DRs and three decoys at tumor cell membrane. This protein has gathered intense interest as a potential candidate for cancer therapy because as a trimmer it selectively induces apoptosis in many transformed cells but not in normal cells.Expression of TRAIL gene in Pichia pastoris GS115 was studied and the strains with highly expression of TRAIL gene were screened out in this study. Using pGEM-TRAIL as template we designed three pairs of PCR primers to modify soluble TRAIL gene fragment. Using pGEM-TRAIL as template and using SOEing technique, we successfully obtained sTRAIL genes that contain KEX 2 site mutation or ATTTA mutation or both of the two. These genes were successfully cloned to the E.coli DH5a to amplify.The modified genes were cloned to the YIP expression vector of Pichia pastoris. After screening with Zeocin, identifying with Xba I IXho I digestion, we obtained recombinant expression vectors: pPICZα-A-TRAILunmodified, pPICZα-A-TRAILKEX2 mutation,pPICZα-A-TRAILATTTA mutations pPICZα-A-TRAILKA double mutation-The recombinant expression vectors were transformed to strain Pichia pastoris GS115 by electrical transformation. After screening with Zeocin and PCR, we obtained recombinant strains: pPICZα-A-TRAILunmodified/GS115, pPICZα-A-TRAILKEX2 mutation/GS115, pPICZα-A-TRAILATTTAmutation/GS 115, pPICZα-A-TRAILKAdouble mutation/GS 115.We confirmed that the optimal pH value was from 5.5 to 6.5, the optimal methanol inducing concentration was 1.0% and the optimal inducing cycles was 96 hours.The recombinant strains of highly expression were isolated by SDS-PAGE. The expression level of pPICZα-A-TRAILunmodified/GS115, pPICZα-A-TRAILKEx2 mutation /GS115,... |
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