Belmont SURFS

Establishing a Biochemical System for the Purification and ATPase activity of GST-Dbp5

The export of mRNA out of the nucleus is a crucial step for the central dogma of eukaryotic life. The export of mRNA transcripts is aided by Mex67, which allows export through the nuclear pore complex doorways in the nuclear envelope. Once out of the nucleus, a protein known as Dbp5, bound to ATP, Gle1, and Nup42 aids in the directionality of mRNA export by helping remove Mex67 from the mRNA strand. In order to release Dbp5 from mRNA to eventually lead the mRNA strand to translation, Dbp5 hydrolyzes ATP so that it unbinds the mRNA. In my project, I am focusing on the purification of Dbp5 in order to develop ATPase assays accompanied by purified Gle1 and Nup42. This will help in better understanding the effects of Nup42 and Gle1 on Dbp5’s ATPase activity and will allow for future study on Dbp5’s ATPase activity. In order to isolate and purify Dbp5, E.coli cells were transformed with a GST-Dbp5 plasmid that were induced with IPTG to enhance production of GST-Dbp5. GST-Dbp5 was then isolated and purified through affinity chromatography. The result of this procedure was small amounts of isolated Dbp5 with a lack of purity. Small-scale ATPase assays were developed to produce preliminary data for Dbp5. A large-scale protein purification of GST-Dbp5 was started and should be followed up with for future experiments.