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 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. Previous efforts worked towards the purification of Dbp5, but the attempts were unsuccessful due to low expression of recombinant protein in E.coli. In this project, I am focusing on enhancing the bacterial induction in order to establish robust purification of recombinant Dbp5. This will help in developing ATPase assays involving Dbp5, Nup42, and Gle1. These ATPase assays will aid 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 enhance the bacterial induction, E. coli cells were transformed with a GST-Dbp5 plasmid and were induced with varying amounts of IPTG to determine the best procedure for bacterial induction. Results from the bacterial induction have indicated that alternative methods for bacterial induction should be explored. Future experiments will look into further enhancing the bacterial induction of Dbp5 in order to establish a biochemical system analyzing the ATPase activity of GST-Dbp5

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

The export of mRNA out of the nucleus, the site of its production, and to the cytoplasm, where proteins are translated, is a crucial step for eukaryotic gene expression. This is aided by Mex67 binding to the transcript, which allows export through the nuclear pore complexes (NPCs), selective doorways embedded in the nuclear envelope. Once the transcript reaches the cytoplasm, a protein known as Dbp5, bound to ATP, Gle1, and Nup42 aids in the directionality of mRNA export by binding to the mRNA strand and removing Mex67. Following interaction with RNA, Dbp5 then hydrolyzes ATP so that it unbinds the mRNA, allowing for enzyme recycling. Therefore, Dbp5 ATPase activity is essential for mRNA export and cell viability. Factors that influence this activity can be accessed through biochemical purification and in vitro analysis. The goal of this work has been to enhance purification of recombinant Dbp5 for this analysis. Specifically, I have focused on optimizing induction of GST-Dbp5 in E. coli Rosetta cells. These efforts have yielded successful induction of Dbp5 at 16°C, and ongoing experiments are aimed at biochemical purification from induced cells. Future experiments will work towards establishing a biochemical system to analyze the ATPase activity of GST-Dbp5