Author: Kim Lange
Approved: Fall 2018
The PurF protein, phosphoribosylpyrophosphate amidotransferase, plays an important role in the formation of purines used by cells. Many crenarchaeal species seemingly have two genes for two similar PurF proteins, but the reason for this redundancy is unknown. The focus of this project was to produce and study the PurF protein from one crenarchaea, Saccharolobus solfataricus. Additional proteins, PurF from E. coli and PurD from S. solfataricus were also produced for later use in the analysis of the SSO PurF. Both PurF proteins were fused to a maltose binding protein (MBP) and histidine tag, respectively, so site-directed mutagenesis was completed to add an enterokinase recognition site so the tags can be later removed. Each protein was produced through induction, extracted through sonication (S. solfataricus) or an inclusion body workup (E. coli), then purified using affinity chromatography. The SSO PurF was further purified through size-exclusion chromatography and successfully digested with enterokinase to remove the MBP. An NMR assay and glutamate dehydrogenase coupling assay were used to determine the activity of the SSO PurF, but neither assay was successful.