Expressing Human Ribosomal Proteins S20 and S26 in Modified E.coli (BL21) Cells

Abstract

Ribosomes are fundamental in creating proteins that contribute to the proper functioning of cellular processes. Thus, ribosome biogenesis must occur with integrity by appropriately expressing ribosomal proteins (RPs). Mutations in the genes of human RPS20 and RPS26 have been linked to disruption in the translation machinery and anomalies in the production of red blood cells, leading to diseases such as Diamond-Blackfan Anemia. To develop our understanding of the role of mutations in human RPS20 and RPS26, we performed ligase-independent cloning of their coding sequence into pNIC28-Bsa4 and pNH-TrxT and transformed these plasmids into a BL21 E. coli expression system. IPTG induction was used to determine conditions necessary to maximize the expression of soluble RPS20 and RPS26. After SDS-PAGE, Coomassie staining showed successful expression of RPS26 in response to IPTG induction, whereas RPS20 was produced regardless of IPTG presence. Human RPs expressed in bacteria are often purified from insoluble inclusion bodies; thus, determining the conditions for maximal solubility provides an optimal strategy for studying fusion proteins. Future research on the effects of the overexpression of RPS20 and RPS26 on human cell growth and viability will expand our understanding of the complex mechanisms of human ribosome biogenesis and its role in disease.