Construction Of Saccharomyces Cerevisiae T7 Expression System Based On Virus Capping Enzyme

T7 expression system has been widely used in prokaryotes because of its strong transcriptional activity and specificity of T7 RNAP.At present,there is no mature expression system of T7 system in yeast cells,because T7 mRNA lacks post-transcriptional processing compared with eukaryotic mRNA.In this study,Saccharomyces cerevisiae was selected as host cell,and the viral capping enzyme was introduced to couple its capping function with the transcription function of T7 system to construct a stable T7 expression system,independent of the host expression system.And by increasing the types of capping enzymes,intein-mediated construction of fusion proteins,and changing the T7 transcription efficiency for exploration and optimization.Contents of present research are indicated as the followings:1.The self-replicating plasmid carrying the stomatitis herpes virus VSV L protein and T7 promoter specifically designed to guide the transcription of EGFP is transferred into the S.cerevisiae strain with integrated T7RNAP in the genome,and the isolated T7 separated from VSV L protein and T7RNAP is initially established Expression system.The results showed that no fluorescence signal was detected,which might be due to the spatial barrier between T7RNAP and capping enzyme or the low capping enzyme activity of VSV L protein.2.In order to eliminate the space barrier between T7RNAP and the capping enzyme,a protein assembly system mediated by Npu DnaE was constructed in Saccharomyces cerevisiae to verify its function.Taking the isolated EGFP as the assembly object,the experimental results show that the assembly efficiency of Npu DnaE-mediated separation of EGFP is 20%.3.To compare the capping activity of the VSV L protein,the RSV L protein of the respiratory syncytial virus,the Kluyveromyces lactis linear plasmid pKGL2 ORF3 protein and the African swine fever virus ASFV NP868R protein were introduced.Using the more sensitive Nanoluc luciferase as the reporter protein,a separate T7 expression system based on four capping enzymes was established.The results showed that the expression level of the system where ASFV NP868R is located is about 10 times that of VSV L protein;then the functionally verified Npu DnaE is introduced into the isolated T7 expression system to mediate the formation of a fusion protein between T7RNAP and the viral capping enzyme.The experimental results show that there is no significant difference between the expression levels of the fusion T7 system and the isolated T7 system;after that,the T7 promoter of the specific design was restored to the complete T7 promoter,and the expression level was still not significantly different.Passage experiments proved that the T7 expression system based on capping enzyme can exist stably in Saccharomyces cerevisiae.Finally,comparing ASFV NP868R with the inducible galactose promoter expression system of Saccharomyces cerevisiae,it is found that the isolated T7 expression system of ASFV NP868R is only about 1/10 of the galactose promoter.4.Transcription efficiency analysis of T7 system in Saccharomyces cerevisiae.The transcriptional efficiency of methionine promoter,galactose promoter,T7 promoter and T7 promoter(deletion of+1 to+3 G)in Saccharomyces cerevisiae was analyzed.It was found that the transcription efficiency of T7 promoter in Saccharomyces cerevisiae was similar to that of methionine promoter.In addition,the deletion of G at+1 to+3 position of T7 promoter reduced the transcription efficiency by 10 times.The transcription level of galactose promoter was about 6.5 times more efficient than that of T7.The transcription level analysis of T7 system in Saccharomyces cerevisiae with free NP868R also confirmed that the transcription efficiency of T7RNAP in Saccharomyces cerevisiae was the key factor affecting the expression level.In this study,we established a stable T7 expression system in Saccharomyces cerevisiae by virus capping mechanism for the first time,and measured the transcription level of T7 expression system in Saccharomyces cerevisiae,paving the way for the further application of T7 system in Saccharomyces cerevisiae protein expression and metabolism regulation.