Abstract 2114 Optimizing the expression, solubility, and purification of human ribosomal proteins SA, S2, S7, and S12 in E. coli to analyze post-translational modifications in vitro

Human ribosomal proteins (RPs) are recognized for their intricate posttranslational modification (PTM) profiles, including phosphorylation, acetylation, ubiquitination, and O-GlcNAcylation, among many others. The dynamic interplay of these PTMs at multiple target residues on individual proteins, responsive to cellular and environmental cues, poses a challenge in deciphering their specific functional roles. To address this complexity and facilitate in vitro studies, we investigated the expression and purification of unmodified forms of four key human RPs-RPSA, RPS2, RPS7, and RPS12-using Escherichia coli as the heterologous expression system. Traditionally hindered by the intrinsic insolubility of these proteins, recent advances have been made by incorporating soluble protein tags. Leveraging ligase-independent cloning, we inserted the coding sequences of RPs SA, S2, S7, and S12 into the bacterial expression plasmid pNH-TrxT, yielding fusion proteins with an N-terminal 6xHis-Thioredoxin A tag. We extended our studies to small-scale protein expression trials conducted under varying conditions of Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, temperature (18, 25, or 37 degrees Celsius, with IPTG concentrations at 0.1, 0.5, and 1 mM), and pre-induction cell density to optimize RP expression. Validation through SDS-PAGE, Coomassie staining, and Western blot analyses unequivocally confirmed substantial post-induction protein abundance for all examined RPs. We proceeded to scale up these tests with larger 50 mL cultures cultivated to facilitate subsequent affinity chromatography purification. Preliminary results show the successful purification of human RPS12, achieved through Nickel resin followed by dialysis. Concurrently, our experiments extended to the expression and purification of both wild-type and catalytically inactive O-GlcNAc Transferase (OGT) fusion proteins. This strategic approach aligns with our overarching goal of conducting in vitro O-GlcNAc labeling experiments, utilizing unmodified RPs as substrates. Looking forward, we will employ in vitro-modified RPs for comprehensive mapping of potential target residues via mass spectrometry and mutational analysis. This foundational work sets the stage for subsequent structural studies and investigations into protein-protein interactions. Our approach aims to unravel the nuanced role of RP modification within the dynamic interior of living cells. Financial support for this project was provided by The First-Year Innovation and Research Experience program at the University of Maryland.