Studies on the functions of human C-terminal tensin-like (CTEN) protein in the nucleus

• Main Authors: Wei Chuang, 莊惟
• Other Authors: Yi-Chun Liao
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/85649669016787421289

碩士 === 國立臺灣大學 === 生化科技學系 === 103 === C-terminal tensin-like (CTEN) protein is localized at focal adhesion complex. It is a member of the tensin protein family, and also plays a crucial role in cell adhesion, migration, and the development of malignancies. Previous studies have indicated that the expression levels of CTEN is elevated in colon cancer, accompanied with the detection of high population of CTEN in the nucleus. It prompts us to study the functions of human CTEN protein in the nucleus. Some of focal adhesion proteins are able to enter the nucleus and function as coregulators or coactivators. Therefore, we hypothesize that CTEN is a DNA binding protein, and could enter the nucleus in cancer cells to regulate the gene expression contributing to tumorigenicity. In this work, we firstly use the online database to analyze the amino acid sequence and the protein secondary structure of CTEN. It turns out that no highly-conserved DNA binding domain is found. On the other hand, we want to determine whether CTEN possesses the ability to bind DNA. First, the recombinant plasmid pET-28a-CTEN, which can express CTEN in E.coli, is successfully constructed. Then we plan to use the purified recombinant CTEN protein to examine its ability to bind DNA. However, high levels of the recombinant protein form inclusion bodies, and the yield of soluble CTEN is low. Moreover, ChIP assay followed by capillary electrophoresis is used to analyze whether CTEN is able to bind DNA. Our results show that the amount of DNA after ChIP is increased in the cells expressing CTEN protein. This result supports the hypothesis that CTEN functions as a DNA binding protein, and provides one potential explanation for the accumulation of CTEN in the nucleus in cancer cells.