Co-expression of the RPS6KB1 and PDPK1 genes for production of activated p70S6K1 using Bac-to-Bac baculovirus expression system

Ribosomal protein S6 kinase 1 (p70S6K1) is a member of the AGC family of serine/threonine kinases and is implicated in a diverse range of cellular processes, including protein synthesis, cell growth, and survival. Dysregulation of p70S6K1, characterized by its overexpression and/or overactivation, has been widely implicated in various human pathologies, particularly in several types of cancer. Thus, the generation of active and recombinant p70S6K1 is critical for investigating its role in cancer biology and for developing novel diagnostic or therapeutic approaches. Here, we report a reliable and efficient methodology for the expression and purification of highly active p70S6K1 (His-actS6K1) in quantity and quality that is suitable for biochemical studies and high-throughput enzymatic assays. To achieve this, we utilized the baculovirus dual expression system, which enabled the co-expression of two recombinant proteins in infected cells: a) His-tagged S6K1 with a deletion of the C-terminal autoinhibitory motif and a phosphomimetic mutation at the mTORC1 phosphorylation site (T389D); and b) untagged PDPK1 lacking the PH domain. Efficient expression of both recombinant proteins was achieved, resulting in highly pure preparations of His-actS6K1. The high activity of the purified kinase was confirmed by various kinase assays, demonstrating significantly higher levels of substrate phosphorylation compared to the tested commercial product. Overall, our developed methodology offers a rapid and cost-effective approach for producing constitutively active His-actS6K1, which can be utilized in academic research and biotechnology.