Generation of a Yeast Overexpression Plasmid for Purification of The Iron Transcription Factor AFT2

Iron is central in many essential intracellular processes, and as a result, balancing its level is crucial. Over two billion people suffer from iron deficiency, decreasing their overall health and leading to illnesses such as anemia. Conversely, too much iron can lead to diseases such as hemochromatosis. Understanding the molecular mechanics of iron homeostasis is the key to alleviating many of the diseases associated with improper iron regulation. Using the model eukaryote S. cerevisiae, many of the basic pathways in iron homeostasis have been uncovered. In yeast, iron absorption and accumulation is regulated by the Aft1 and Aft2 transcription factors that control the expression of the genes collectively termed the iron regulon. In low-iron conditions, the Aft2 factor activates gene expression to increase intracellular iron levels. To better understand the molecular mechanism used by Aft2 to regulate gene expression, attempts have been made to express and purify the full-length Aft2 protein in E. coli for biochemical analysis. This problem was addressed through the successful construction of an expression vector containing the AFT2 full-length gene using the Gibson Assembly protocol. This new expression plasmid will allow the expression and purification of Aft2 directly from yeast. Large amounts of the purified protein will enable exploration of the DNA and iron-binding properties of this iron-responsive transcription factor at the molecular level