Abstract 2329 Development of a universal expression and purification system for human ribonuclease inhibitor (RNH1)

The Human Ribonuclease Inhibitor (RNH1) is an essential mammalian protein with a complex horseshoe-shaped structure and a broad range of functions including ribonuclease (RNase) A inhibition, transcriptional and translational control, and oxidative stress responses.The structure of RNH1 features leucine-rich-repeats (LRRs) and a remarkably high portion (7%) of reduced cysteine residues, which upon oxidation leads to structural collapse.The long-term goal of this work is to construct variants of RNH1 where oxidation-sensitive cysteines are replaced by leucine, thereby enabling us to dissect the role of oxidation sensitivity from the structural stability and ribonuclease binding activity of the protein.RNH1 is notoriously difficult to express and purify.Prokaryotic expression systems suffer from low yield and purification strategies either rely on the RNase A affinity of RNH1 or an affinity tag incompatible with ideal reducing conditions.We have addressed protein expression challenges in E. coli by assessing soluble protein production in cultures grown with DTT, coexpression of GroEL/ES, and enhanced tRNA production with pRARE.To maximize protein yield for in vitro studies, we are also assessing purification yield using an N-terminal GST-tag, 2x-Strep tag, or the C-terminal C-tag.I will present and compare protein yields between each system described above to determine an optimal expression and purification scheme for RNH1 and its variants.