hRPL38 Expression & Purification for Biophysical & Post-Translational Analysis

Abstract

Although the overall structure and function of the human ribosome are well characterized, less is known about the roles of individual ribosomal proteins that compose it. Increasing evidence suggests that many ribosomal proteins have extraribosomal functions, contributing to processes such as development, immune signaling, and disease¹,². Ribosomal protein L38 (RPL38/eL38) is a component of the 60S ribosomal subunit and has been shown to selectively regulate translation of Homeobox (Hox) mRNAs, playing a critical role in vertebrate tissue patterning¹. Disruption of RPL38 leads to axial skeletal defects and has also been implicated in cancer-associated translational control¹,³. Additionally, RPL38 undergoes multiple post-translational modifications, though the functional significance of these modifications remains largely unexplored4. This project aimed to optimize bacterial expression and purification of human RPL38. The RPL38 gene was cloned into a pNH-TrxT vector and transformed into E. coli. Expression trials tested varying IPTG concentrations (0–0.1 mM) and temperatures (18–37 °C), with SDS-PAGE analysis showing maximal expression at 37 °C with 0.1 mM IPTG. Western blotting confirmed that the observed band corresponded to the 6×His-TrxT-RPL38 fusion protein. For purification, cells were lysed via sonication or glass bead disruption, both yielding comparable protein recovery. RPL38 was present in the soluble fraction at levels sufficient for downstream purification. Initial Ni-NTA affinity purification via spin columns did not recover protein in elution fractions; however, modifications to buffer conditions and use of Ni-NTA agarose resin resulted in detectable RPL38 in the elution. AlphaFold structural predictions indicate that the His-tag is likely accessible, suggesting that purification inefficiencies are due to experimental conditions rather than tag occlusion. Since RPL38 was successfully detected in the elution, future work will focus on optimizing purification conditions and scaling up protein production to obtain sufficient quantities for post-translational modification assays and protein–protein interaction tests with HEK293 lysate.