| People pay more and more attention to biological safety, especially in the aspects of food safety. Genetic engineering technology is bringing convenience to people, at the same time it is also facing more and more challenges. Schizosaccharomyces pombe is similar to higher organisms in biological characteristics. But the Schizosaccharomyces pombe expression system has been ignored for a long time. The reason is because the Schizosaccharomyces pombe expression vector is the main limited factor. Schizosaccharomyces pombe using as host cells in our laboratory, our goal is to construct high efficient expression vector with biological safety. We acquired the target mainly through the arificial optimization design of expressing vector.1. Seeking the high expression housekeeping gene of the Schizosaccharomyces pombe. Through the intracellular protein SDS-PAGE analysis of different growth stage of the Schizosaccharomyces pombe, we discovered that a protein has a higher expression in each stage. By traditional ammonium sulfate fractionation precipitation method, we isolated the high-abundance protein. Through the analysis of the protein N terminal amino acid sequence, the high-abundance protein is finally identified to gene enolase of Schizosaccharomyces pombe. The protein consists of 439 amino acids, with predicted molecular weight of 47.3 kD. We guess that promoter might be a strong constitutive promoter.2. Researching the transcription activition intensity of two kinds of constitutive promoters. Through assaying the LacZ enzyme activity under the control of the promoter sequence control of different length fragments, we determined the eno101 promoter length is 276 bp, the gpd3 promoter length is 800 bp. At the same time, we also found that only the promoter region of CpG island and the core promoter sequences exist at the same time, in order to maintain the housekeeping gene high expression. If the lack of CpG Island, promoter can still start transcription of target gene transcription, but the strength will be weakened. At the same time, we will also compare the two kinds of promoter and nmtl promoter for researching the transcriptional effects of the two promoters. By assaying the LacZ enzyme activity, transcription strength of eno101 and gpd3 promoter is similar, and it is 1.5 times more than the transcriptional efficiency of nmt1 promoter. This indicates that these two kinds of promoter in Schizosaccharomyces pombe belongs to the strong promoter, so that it brings the important value to the construction of our biological safety expression system.3. Artificial design and optimization of promoter from biological safty selectable marker gene. On the basis of the promoter sequences derived from Schizosaccharomyces pombe gfal gene, artificial design policy is appllied to integrate the Escherichia coli σ70 core promoter sequences to it. At the same time, the ribosome binding site sequences are joined. On the basis of shuttle vector pREP3X, the plasmid pGFA-nmt was constructed. Plasmid pGFA-nmt included a selection marker gene gfa used both in E. coli⊿glmS and S. pombe⊿gfal, but not containing amp and leu gene, and reached to the biological security screening purposes. Plasmid pGFA-nmt length is 7882 bp,900 bp less than the original pREP3X with 8784 bp, and achieved the expected goal.4. Successfully constructing two kinds of plasmids containing eno101 and gpd3 promoter respectively belong to biological safety expression vector of Schizosaccharomyces pombe. The vector can take function in Escherichia coli ⊿glmS and Schizosaccharomyces pombe ⊿ gfal with the same selected marker gfa, no longer containing other selected marker gene. Xylanase gene xynA2 as a reporter gene was used to verify the function of the new constructed plasmid. In the culture solution of engineered S. pombe haboring the newly constructed plasmids pGFA-eno-CX and pGFA-gpd-CX, xylanase XynA2 in YES and EMM medium was produced effectively, and extracellular xylanase activity reached 30 U/ml.
|
PDF Full Text Request