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

Abstract

Background: Ribosomal protein S6 kinase 1 (p70S6K1) is a member of the AGC family of serine/threonine kinases which plays a role in various cellular processes, including protein synthesis, cell growth, and survival. Dysregulation of p70S6K1, characterized by its overexpression and/or hyperactivation, has been implicated in numerous human pathologies, particularly in several types of cancer. Therefore, generating active, recombinant p70S6K1 is critical for investigating its role in cancer biology and for developing novel diagnostic or therapeutic approaches.

Methods: The baculovirus dual expression system was utilized, enabling 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. The high activity of the purified kinase was confirmed by immunoblotting, as well as by Kinase-Glo and AlphaScreen kinase assays.

Results: Efficient expression of both recombinant proteins was achieved, resulting in highly pure preparations of His-tagged p70S6K1. The high activity of the purified kinase was confirmed through multiple kinase assays, demonstrating significantly higher levels of substrate phosphorylation compared to the tested commercial product.

Conclusion: 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/biophysical studies and high-throughput enzymatic assays. 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.

Keywords: Baculovirus expression system; Kinase activity; PDPK1; Protein expression; Protein phosphorylation; S6K1.

Fig. 1

Schematic diagram of recombinant baculovirus construction. A) Domain structure of wild type and recombinant PDPK1 and p70S6K1; B) Construction scheme of the recombinant plasmid co-expressing modified p70S6K1 along with PDPK1 in the pFastBac™ Dual vector

Fig. 2

His-actS6K1 was efficiently expressed from the recombinant baculovirus and phosphorylated at the PDPK1-specific site T229. A) SDS-PAGE of purified His-actS6K1 protein stained with Coomassie InstantBlue. The samples from the final dialysis of three preparative expressions/purifications were separated on the gel; B) Western blot analysis of purified His-actS6K1 T229 phosphorylation using the phospho-T229 antibody. 1 – lysate (20 µg) of non-infected Sf9 cells; 2 – lysate (20 µg) of Sf9 cells infected with recombinant His-actS6K1 baculovirus; 3 – purified His-actS6K1 (1 µg). Positions and values of the protein marker are applicable for both A) and B)

Fig. 3

His-actS6K1 enzymatic activity was characterized using different substrates and assay platforms. A) Phosphorylation of recombinant GST-rpS6 protein tested with the phospho-rpS6 (S235/236) antibody; B) Kinase activity of His-S6K1 preparations and commercial S6K1 were tested in the serial 1:5 dilutions using the same substrate and phospho-rpS6 (S240/244) antibody C) Kinase activity in the presence (black circles) or absence (open circles) of the rpS6 substrate peptide (synthetic peptide Biotin-QEQIAKRRRLSSLRASTSKSESSQK) tested using the Kinase-Glo assay

Fig. 4

Inhibition of the His-actS6K1 catalytic activity by the pan-kinase inhibitor Staurosporine (A) and the S6K1-selective inhibitor LY2584702 (B) tested using the AlphaScreen assay. The results were obtained from four independent experiments conducted in triplicate. Dose-response curves and statistical analysis were performed using GraphPad Prism 9