Sciences and Mathematics, College of
Chemistry and Physics, Department of
SURS Faculty Advisor
Rachel E. Rigsby, PhD
The export of mRNA out of the nucleus is a crucial step for eukaryotic gene expression. 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. Following interaction with RNA, Dbp5 then hydrolyzes ATP so that it unbinds the mRNA, allowing for enzyme recycling. The goal of this study is to explore the ATPase activity of Saccharomyces cerevisiae Dbp5 in vitro with purified recombinant protein. Previous efforts worked towards enhancing the bacterial induction of Dbp5 with the Rosetta strain of the E. coli. This semester, I aimed to optimize the purification of Dpb5 from cells via affinity chromatography. To purify Dbp5, Rosetta cells were transformed with a GST-Dbp5 plasmid and induced with IPTG at 16 °C. To ensure successful induction, the bacterial cells were sonicated and the purification process was started. Future experiments will work towards further optimizing and refining the purification of Dbp5 in order to establish a biochemical system analyzing the ATPase activity of GST-Dbp5. This will help in developing ATPase assays involving Dbp5, Nup42, and Gle1.
Utley, Sarah; Rigsby, Rachel E. PhD; and Adams, Rebecca L. PhD, "Establishing a Biochemical System for the Purification and ATPase activity of GST-Dbp5" (2023). Science University Research Symposium (SURS). 66.