Apoptosis Inhibitory Factor Of Survivin Gene And Its Expression In Pichia Pastoris

Objectives: 1.To establish and use the eukaryotic (Pichia pastoris) expression system for expressing survivin protein—an inhibitor of apoptosis proteins. Methods: 1.Full length Survivin gene was amplified from recombinant plasmid pGEX-4T-3-HA-Survivin by using survivin specific primers. Then, Survivin gene was inserted into pGEM-T, and verified by sequencing. 2.Verified Survivin gene was subcloned into pPic9k. The recombinant plasmid was linearized by restriction enzyme cutting. The linear Survivin gene was introduced into HIS-/GS115 cells by electroporation. The transformants were transferred onto YPD plates that contained different concentrations of G418 for selecting the positive clones. The integrated Survivin gene in positive clones was confirmed by PCR. 3.Selected transformants were cultured in BMMY medium with 1% methanol for inducing the expression of survivin protein. Survivin protein expression was analyzed by ELISA. Results: 1.The result of sequencing human Survivin compared with the Survivin sequence in the Gen-Bank is the same. 2. We have made up an eukaryotic (Picha pastoris) expressive vector(pPic9k-Survivin). After the linear recombinant vector was introduced into HIS-/GS115 cells, we have selected 6 positive clones against G418(4mg/ml)and confirmed by PCR. 3. Suvivin protein reached highest concentration when expressed for two days in 1% methanol BMMY medium. Conclusions: To express survivin protein in Pichia pastoris is possible. Improved survivin protein yield may be reached by modifying the experimental conditions. This is necessary for tumor diagnosis, anticancer therapy, and the fundamental research of tumor pathology.