Establishing phosphorylated human protein kinase N3 generation via PDPK1 Co-expression in Sf9 cells

Abstract

Protein Kinase N3 (PKN3) is an AGC family serine/threonine kinase implicated in cancer due to its elevated expression in tumour cells. Its activation requires phosphorylation (Thr718) and regulation by upstream factors, notably by phosphoinositide-dependent kinase 1 (PDPK1), which increases the amount of active PKN3. This work utilises the MultiBac baculovirus system to co-express PKN3 and PDPK1 in Sf9 cells, enabling production of phosphorylated PKN3. The protein is purified via Strep-tag affinity chromatography using Strep-Tactin. Furthermore, immobilisation of PKN3 on magnetic beads is explored to develop fluorescence-based assays, providing a platform for future high-throughput screening of PKN3 inhibitors.