Expression and Purification of Cytotoxic Human Ribosomal Proteins in Cell-Free Systems

Abstract

Ribosomes are the factories of the cell, catalyzing protein synthesis. Human ribosomes are made up of the 40S small subunit and 60S large subunit to form the 80S ribosome. These subunits are composed of about 80 proteins and 4 rRNAs. Some proteins, including RPS6/eS6 and RPS27/eS27, are difficult to express in standard inducible bacterial whole-cell cultures due to their charge profiles, tendency to form inclusion bodies, and cytotoxicity to bacterial cells. The use of bacterial cell-free expression systems (CFES) offers a promising approach for synthesizing challenging human ribosomal proteins, given their tolerance to toxic effects. In past experiments, MyTXTL, a commercial CFES, has been used to validate the expression of soluble ribosomal proteins, such as RPS10/eS10 and RPS12/eS12, as well as RPS27/eS27, which is toxic when expressed in intact cells. We used a validated in-house CFES previously prepared in our laboratory, along with the IPTG-inducible pNH-TrxT bacterial expression cloning vector, to express human ribosomal protein-coding sequences in overnight cell-free reactions at 37ºC with shaking. Purifications were performed via affinity chromatography with Ni-NTA spin columns, and protein abundance was evaluated using SDS-PAGE and Western Blot analysis. We successfully expressed and purified RPS6/eS6 from the pNH-TrxT-RPS6. These results validate the use of our in-house CFES system as comparable to the commercially available system and bacterial lysates from other academic laboratories. Furthermore, we can use this system to express and purify additional ribosomal proteins that have previously demonstrated cytotoxicity in whole-cell systems to investigate the role of post-translational modifications, such as O-GlcNAcylation, in ribosomal protein function.