Fusion Expression In Pichia Pastoris Of Bovine Interon-tau Gene Mature Protein Coden Region And Purification Of Expressed Protein

Bovine interferon-tau(bIFN-Ï„) which is from trophoblast of early embryo could promot corpus luteum development,interrupted the process of antiluteolytic mechanism in bovine endometrial,and finally cause the beginning of gestation.Many studies showed: bIFN-Ï„played crucial role in pregnancy recognition and regulated the transition process from pregnancy recognition to embryo implantation.In this study,coding sequence(CDS) of mature interferon-Ï„(bIFN-Ï„) gene from Chinese Hostein was cloned from a prokaryotic expression combinant(pET-28a-bIFN-Ï„). The sequence added His₆-tag in its C-terminal was named bIFN-Ï„-His₆.The complete ORF was discovered and considered as a bIFN-Ï„gene by BLASTN and ORF Finder.The full gene was 548bp in length,and it coded a 182 amino acid multipeptide.The results indicated that bIFN-Ï„-His₆ had the specialized features of the encoded protein,including N-glycosylation sites,codon bias,secondary stricture and hydrophobicity predicted and analyzed.The introduction of His₆-tag increases the codon preferences between Pichia pastoris and the gene.The difference can affect the expression in Pichia pastoris of the gene.In our studies,we used pPIC9K as expression vector and GS115 as host strains to express bIFN-Ï„-His₆ protein.After the bIFN-Ï„-His₆ gene with EcoRâ… and Notâ… restriction Sites and His₆-tag was cloned to pMD18-T vector,pMD18-T-bIFN-Ï„-His₆ and pPIC9K were cutted by EcoRâ… and Notâ… and the cutted fragments were ligated by T4 DNA ligase. The bIFN-Ï„-His₆ gene expression vector,pPIC9K-bIFN-Ï„-His₆,was constructed successfully after sequencing.The expression vector pPIC9K-bIFN-Ï„-His₆ used for transformation,was linearized by Sac I.Pichia pastoris GS115 strains were made competent and transformed with Sal I-linearized pPIC9K-bIFN-Ï„-His₆ by electroporation. After selection by MM plates and MD plates,YPD plates containing G418 and PCR,some positive colonies which exhibited different anti-G418-levels were obtained and the results of identification showed that the transformants were Mut⁺ and positive by PCR.The recombinants of Pichia pastoris were then induced by methanol and the maximum products were emerged when they were induced for 96h.The bIFN-Ï„-His₆ protein expressed in Pichia pastoris approximately 25kD was purifed by affinity chromatograph used HisTrap^TM HP and BioLogic DuoFlow.Purified bIFN-Ï„-His₆ expression products were analyzed by SDS-PAGE and found a major protein band at a molecular weight of 25KDa which was expressed successfully.