Cloning of Human Suv39h1 for Inhibition Studies

Abstract

Cancer is one of the leading causes of death in the Western world and is characterized by abnormal cell growth. Despite the advancement in genetic engineering, there is not a significant amount of change in the rate of morbidity associated with the disease. For human colorectal cancers (CRC), early diagnosis and treatment is important for survival; if the cancer has metasta- sized to the liver, the survival rates are poor. Hence, improvements must be made to the existing therapies for metastatic human CRC. Applying knowledge of molecular mechanisms to modify and control the outgrowth of cells is the key to introducing new therapies. Fas is a receptor protein involved in apoptosis or, more commonly known as, “cell death.” Human carcinoma cells lower the expression of Fas on the cell surface. This lowering of expression levels of Fas is correlated with increased levels of histone H3 lysine 9 trimethylation (H3K9me). SUV39H1 is a histone lysine methyltransferase which catalyzes H3K9me. The goal of this study is to identify novel SUV39H1-specific inhibitors to decrease the level of methylation at the Fas promoter. Thus, to initiate our study, we have cloned the gene for human SUV39H1 into the expression vector pET-21c(+). Upon transforming the recombinant DNA into an expression strain of E. coli such as BL21, we will overexpress and purify SUV39H1 using affinity chromatography. Ultimately, the activity of SUV39H1 will be tested in the presence of various inhibitors.