Expression, Purification And Biological Activity Analysis Of Recombinant Human Soluble TRAIL In Pichia Pastoris

Optimization of fermentation conditions, protein purification craft and assay of biological activities of recombinant human soluble TNF梤elated apoptosis inducing ligand (TRAIL) were studied in this research. Meanwhile, the tertiary structure of TRAIL was predicted by using biological soft wares and Internet.Cultures in shaking bottle showed the optimum conditions as follows: the culture medium is BMGY/BMMY; the pH is 5.5-6.5; methanol concentration is 1%; inducement period is 96h. High-density fermentation in 5L fermentor was further studied. High-density fermentation craft of TRAIL were determined with fed-batch fermentation.The supernatants of fermentation were treated with 30% - 60% (NH4)2SO4 to gradiently precipitate the proteins followed by desalination through a Sephadex G25 gel colume. The desalted proteins were purified by Q-Sepharose-Fast Flow ion exchange chromatography and Sephadex G50 fine gel filtration. The purity of target protein-rhsTRAIL was as high as 95%. SDS-PAGE electrophoresis showed the molecular weights of monomer and dimmer based on were 22,000Da and 44,000Da, respectively. The iso-electric point of was 4.8-5.2. The amino acid analysis indicated that the recombinant protein is non- N-glycosylated.The ability of 0.5 ug/m rhsTRAIL to induce apoptosis of liver cancer cells SMMC7721 and BEL 7402 could be visioned morphologically. This was further verified by Electrical microscope observision of induced SMMC7721 cells and agarose gel electrophoresis of apoptosis DNA. MTT test results also showed that 0.01 u g/ml purified rhsTRAIL has strong effect to induce apoptosis of SMMC7721 cell.Antheprot software and Internet were used to predict secondary and 3D structure of recombinant TRAIL segment (114aa-281aa). Mixing type structure was showed. The edextracelluar C segment (114aa-281aa) formed eight paralleled 3 -folding typical 3 sandwich structure domain.