| Summary: | 碩士 === 國立臺灣師範大學 === 生命科學研究所 === 97 === Abstract
Many neurodegenerative diseases are linked to abnormally expanded CAG repeats in the coding regions of responsible genes. One of them, spinocerebellar ataxia type 17 (SCA 17) was identified with CAG trinucleotide repeat expansion in the TATA-box binding protein (TBP) gene on chromosome 6q27. The possible molecular pathogenic mechanisms of SCA17 could be aggregation caused by mutant TBP with expanded polyglutamine strentches and dysfuction of transcriptional regulation by loss of function. Although the detail pathogenic mechanism is unknown in poly-Q disease, inhibition of aggregation is effective to protect cell in vitro and in vivo. In this study, an E. coli. expression system was established to express the TBP-Lac Zα fusion proteins. By structural α complementation of β Galactosidase (β Gal), we anticipate this system could be a bacterium-based model for rapid drug screening to inhibit protein aggregation. In plasmid construction, we used pGEX plasmid to construct several protein expression vectors including those harboring TBP-Lac Zα fusion genes with various length of polyQ tract and pGEX-Lac Zα. In protein expression, several E. coli. strains were tested to express these recombinant proteins by blue-white screening, and expression conditions were obtained to express soluble short poly-Q TBP as blue color culture, and aggregated long poly-Q TBP as white color culture. We validated this drug screening system by phenotypic screening, genotyping, SDS-PAGE, Western blotting and β gal activity assay. The result showed that JM109 E. coli. expression system can express pGEX-Lac Zα as blue color culture and pGEX-fTBP40Q-Lac Zα as white color culture. We used pGEX-fTBP40Q-Lac Zα/JM109 as drug screening model. In drug screening, several candidate drugs capable of inhibiting poly-Q protein aggregation (such as trehalose and congo red) were tested to prevent TBP aggregation. The results show that trehalose and congo red can not inhibit long poly-Q TBP aggregation by blue-white screening. This study could provide a high-throughput microbial drug screening system.
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