Active Molecules In Of Methanol Pichia Yeast Pichia The Pastoris And The Expression Of Specific Dna-protein Binding Factor Purification And Identification

Pichia pastoris gene expression system is a second generation expression system in yeast after the S.cerevisiae. It has not only many advantages of the prokaryotic expression system such as growing quickly,manipulating easily,strong power to express and control foreign protein,but also the ability to make foreign protein modified in post-translation. So Pichia pastoris gene expression system is more appropriate to express many eukoryotic proteins than prokaryotic system and has attracted more and more people' s attention on it. It has been used to express in an attractive level a variety of intracellular and extracellular proteins of interest.ln our study,Pichia pastoris gene expression system was used to express the soluble part of human TRAIL (TNF Related Apoptosis Induced Ligand) and antibacterial peptide-CEME.Human TRAIL is a new member of TNF superfamily,which can specially induce cancer cell to apoptosis,but has none effect on normal cell. It is a new potential anti-cancer drug. The soluble functional part of TRAIL gene was modified based on the partial principle code found in Pichia pastoris. It was then cloned to the secreted vector-pPIC9K and recombined successfully into the chromosome of Pichia pastoris host strain-Gsl 15 by electroporation. After inducement by methanol,the soluble part of TRAIL was secreted into supernatant. A high-level strain named as GTr48 with more than 38% of total protein secreted was screened. The foreign protein expressed amount to 10mg/L by estimate. The supernatant of GTr48 can restrain the growth of cancer cell,as compared with the supernatant of wild-type after inducement. The result shows that the soluble part of human TRAIL expressed by GTr48 have the native function of TRAIL.Antibacterial peptide is a kind of small peptide found in animals and plants and has a broad-spectrum antibacterial activity. It play a key part in the immune system. It could recognize bacterial membrane and then bind it to form holes which cause the content of cell leaked out and finally the death of the bacteria. Antibacterial peptide can take the place of antibiotics which easily lead to anti-drug activity of bacteria. It has become a highlight on the study of anti-bacteria now. Because of its special function,no successful example evolving the expression of antibacterial peptide directly in the prokaryotic system havs bben reported until now. The artificial antibacterial peptide-CEME gene was designed according to the codon with the highest frequency in Pichia pastoris,then the CEME gene was integrated into the chromosome of Pichia pastoris strain-Gsl 15 by electroporation. After optimizing the condition of inducement,three strains displayed obvious antibacterial activity were obtained. Staphylococcus auresu and Hay bacillu were killed by it but the control was not. So these strains can be expected to become an additive for resistiong disease caused by bacterial in aquaculture.