High-Throughput Microorganism-Based Screening of Novel Molecules for SCA 17 Treatment: Monitoring TBP solubility by chloramphenicol resistance

• Main Authors: Zi-Ling Yong, 永資陵
• Other Authors: Guan-Chiun Lee
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/84795821833052309319

碩士 === 國立臺灣師範大學 === 生命科學研究所 === 98 === 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 trinucleo- tide repeat expansion in the TATA-box binding protein (TBP) gene on chromosome 6q27. The possible molecular pathogenic mechanisms in 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, we establish an E. coli. expression system to express the TBP-chloramphenicol acetyltransferase (CAT) fusion proteins. We constructed a simple in vivo system for assessing protein solubility that involves expressing a fusion of an expanded polyQ TBP with CAT, the enzyme responsible for conferring bacterial resistance to chloramphenicol. In plasmid construction, we used pGEX and pET plasmid to construct several protein expression vectors including those harboring TBP-CAT fusion genes with various length of polyQ tract、pGEX-CAT and pET-CAT. In protein expression, several E. coli. strains were tested to express these recombinant proteins by Cm resistance, and expression conditions were obtained to express CAT fuses with insoluble TBP-Qn, they confer significantly lower or even no Cm resistance, and vice versa. We validated this drug screening system by phenotypic screening, genotyping, Western blotting and CAT assay. The result showed that Tuner and BL21(DE3) E. coli. expression system can express CAT fuses (Tuner/pGEX system、JM109/pGEX system、JM109(DE3)/pGEX system and BL21(DE3)/pET system) with insoluble TBP-Qn, they confer significantly lower Cm resistance, and vice versa. We used Tuner/pGEX-tTBP60Q-CAT and BL21(DE3)/pET-tTBP60Q-CAT as drug screening model. In model test of positive control, 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 Cm resistance. Maybe trehalose and congo red could not suppress the expression of this system to extend the length of TBP protein aggregation. To prove that this system can be used as the screening model of other drugs, thus reducing the culture temperature, was found poly-Q protein reduced aggregation, and the resistance was increasing. This study could provide a high-throughput microbial drug screening system, and this system can be used as other drug screening models and analysis by phenotype, genotype analysis, Western blot and CAT activity analysis to find inhibit the aggregation of long extension of TBP protein potential drug.