| Summary: | 碩士 === 國立臺灣大學 === 生化科技學系 === 102 === The methylotrophic yeast Pichia pastoris is a generally used expression system in production of industrial and pharmaceutical proteins. Traditionally, screening high-yield candidates relies on assay transformant by transformant. However, It is labor extensive, time consuming and unstable. In this study, a high-throughput screening system in P. pastoris was developed. The polycistronic expression plasmids harboring the genes of Endostatin-IL2, 2A peptides from foot-and-mouth disease virus and EGFP were introduced into P. pastoris. The transformants and wild types were distinguished by their fluorescence. Furthermore, since previous study using prokaryotic protein as target protein, this study replace the protein to the eukaryotic fusion protein with more complex structure. The results showed this system successfully express the eukaryotic fusion protein. There is positive correlation between the production of Endo-IL2 and EGFP. Furthermore, the transformants could be isolated using fluorescent-activated cell sorter (FACS) based on their fluorescence intensity. However, because using the single colony as sample and cultivated to sort, the Endo-IL2 production of transformants sorting by FACS was not better than the original transformants. It still needed to continue study to solve this problem.
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