Belmont SURFS

Purification of MBP-Gle1 and Stimulation of Dbp5 ATPase Activity

The export of mRNA from the nucleus into the cytoplasm is essential for gene expression in eukaryotes such as S. cerevisiae. This process is enabled by a variety of cellular proteins, including Gle1. Gle1 is essential for mRNA export because it enhances the ATPase activity of another protein called Dbp5, which ensures that mRNA remains in the cytoplasm for translation following export. To biochemically explore how Gle1 stimulates Dbp5’s ATPase activity, my objective was to establish a system for the biochemical purification of Gle1. I hypothesized that the addition of purified Gle1 to Dbp5 and another assisting nuclear pore protein, Nup42, would lead to a significant increase in relative Dbp5 ATPase activity. To test this, I induced BL21 E.coli cells to express Gle1 fused to a maltose binding protein tag (MBP), purified MBP-Gle1 through affinity chromatography and dialysis, and assessed relative ATPase activity with spectrophotometry. This biochemical system will allow for further study of the proteins associated with mRNA export at Belmont University.