Screening And Application Evaluation Of Highly Efficient Transcription Terminator In Pichia Pastoris

Methylotrophic Pichia pastoris is a commonly used host for the expression of foreign proteins,and expression cassette elements play an important role in the expression of foreign proteins in Pichia pastoris.The terminator commonly used in Pichia pastoris is AOX1 TT.Whether it is possible to find a better terminator to replace AOX1 TT is important for the expression of exogenous protein in Pichia pastoris.In this study,the important element terminator in the gene expression cassette was used as the research object.By screening the terminator from the endogenous source of Pichia pastoris and using the synthetic terminator of Saccharomyces cerevisiae,the important role of the terminator in the expression of foreign proteins was determined.The effects of other factors to terminator such as promoters and reporter proteins in the expression of foreign proteins were explored through experiments.The specific research contents and results are as follows:(1)In this study,the nucleosome occupancy prediction of Pichia pastoris endogenous transcription terminator was carried out,and the terminator of Saccharomyces cerevisiae was used to replace AOX1 TT used in Pichia pastoris.By measuring the copy number of the recombinant strain,the effect of copy number on the expression of the foreign protein was excluded.(2)By measuring the fluorescence of EGFP,it was found that the fluorescent expression of the recombinant strain was increased,and the fluorescence expression of the 0104 terminator strain was the most increased,and the relative fluorescence of the AOX1 TT was increased by about 65%.In the replacement of the S.cerevisiae synthesis terminator,the relative fluorescence of Tsy3 terminator was increased by about 48%.Prove that the terminators can be applied to enhance the expression of foreign proteins.(3)By quantitative PCR analysis,it was found that the transcription level of the EGFP gene was increased,and the transcription level of the 0104 terminator was the most increased one,which was about 3.7 times that of AOX1 TT.In the replacement of S.cerevisiae synthesis terminator,Tsy3 increased the most,about 2.4 times that of AOX1 TT.Prove that the terminator affects gene expression by affecting the level of gene transcription.(4)The Pichia pastoris strain was replaced by constructing a terminator with different promoters and reporter proteins,and the effect of exogenous protein expression was evaluated.It was found that the foreign protein gene and promoter had a certain effect on the terminator.For the terminator itself,the longer the necessary element sequence length,the more obvious the effect of the terminator on the expression of the foreign protein.This study reveals the important role of the terminator in the expression of foreign proteins in Pichia pastoris,and provides a basic theoretical basis for optimizing the expression of recombinant proteins by expression elements in Pichia pastoris and other eukaryotic cells.