| Summary: | 碩士 === 國立臺北科技大學 === 化學工程與生物科技系生化與生醫工程碩士班 === 107 === Using eukaryotes as an expression system, although not as efficient as prokaryotes, but it has the function of post-translational modification, no endotoxin production, and less concern of inclusion bodies formation. In this study, we’ve chose the yeast Yarrowia lipolytica as the host of protein expression. The aim of this study was to construct a multi-copy integration vector in order to enhances the heterologous protein expression in Yarrowia lipolytica. The multiple repeats of rDNA and Ylt1 were used for homologous recombination. We used 18S rDNA, 26S rDNA, and different size fragments of Ylt1 as the multi-copy integration region to construct recombinant plasmid. The homologous recombination of the reporter gene-red fluorescent protein gene was inserted into the yeast chromosome to observe the expression of the protein, but the plasmids of the different size fragments Ylt1 of 1.5 kb, 3 kb and 5 kb transform into Yarrowia lipolytica were no colony growth. The reason may be that the reverse transcriptase in the CDS region is inhibited by repressor and the gene cannot be expressed. The successful transformant of pYLF5+ Ylt1 repeat and pYLF5+26S rDNA was followed by multiple repetitive transformations, allowing more genes to be inserted into Y. lipolytica. It was found that there was a significant increase of red fluorescent protein expression in the seventh transformation, and q-PCR results show that the number of genes embedded is increased by three to four times, demonstrating that it is feasible to use the repeat sequences in yeast as sites for gene insertion. Finally, we want to construct an integrated plasmid expression system containing heterologous protein and connecting red fluorescent protein DsRed. The expression level of can be determined by the color from fluorescent protein. We expected that the integration plasmid expression system can be applied more widely in the future.
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