Cloning and Expression of Human RPS24 into E.coli and the HEK293 Cell Line

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Ribosome biogenesis is the process of constructing ribosomes and requires ribosomal RNA, ribosomal proteins (RPs), and assembly factors. The products of eukaryotic ribosome biogenesis are the large 60S subunit and the small 40S subunit, of which RPS24 is crucial in its formation. In addition to its translational roles, RPS24 is associated with regulating cell growth and proliferation, apoptosis, and DNA damage response. We hypothesize that the extra-ribosomal functions of RPS24 are impacted by its post-translational modifications (PTMs). To elucidate the functions of these PTMs, the coding sequence of human RPS24 was cloned into pNH-TrxT and pNIC28-Bsa4 bacterial expression vectors via ligase-independent cloning. The recombinant plasmids were then transformed into BL21 E.coli cells, and initial trials were conducted to optimize growth and expression conditions for the two transformed strains. Protein expression was determined using SDS-PAGE and Coomassie staining, the results of which indicated moderate levels of the RPS24 fusion protein in cells transformed with both the recombinant plasmids. RPS24 expression was observed without IPTG induction, indicating leaky expression. In parallel experiments, we investigated the overexpression of RPS24 in HEK293 cells from the plasmid pcDNA3.1(C)DYK, and successful transfection was confirmed by SDS-PAGE and Western Blot analysis. We aim to investigate the role of RPS24 PTMs in cell proliferation and viability under various stress conditions to evaluate their impact on tumor development.