Abstract B123: Full-length bio-active atypical protein kinase c ι expression and purification in a bacterial system

Abstract

Abstract Background: Proteins are at the steering wheel in the route of cancer progression. Protein kinase C (PKC) is a contributor to malignant advancement and it pushes past some of the normal cell’s defenses. In particular, PKC-ι participates in oncogenesis at varying levels in signaling and tissue specificity. Around 70% of the selected chemical entities that are the result of in-silico investigations are found to be rejected in the final stages of drug development. Therefore, binding studies are essential to in-silico techniques and are pivotal for finding kinase inhibitors for overly active proteins like PKC-ι. Despite the fact that there are numerous binding studies involving PKC-ι, protein production remains tedious and expensive. Objective: In this investigation, we hope to produce large amounts of recombinant bio-active full-length PKC-ι in a bacterial system. The large quantity of PKC-ι can be used for future experiments such as Nuclear Magnetic Resonance (NMR), x-ray crystallography, isothermal titration calorimetry (ITC) and ligand binding studies. Methods: We used bacterial expression plasmids (pET-His) with full-length and catalytic domain only PKC-ι gene inserts (tagged and untagged with GST). We transformed the bacterial cells lines BL21(DE3), Tuner (DE3), and ArticExpress. Results and Discussion: So far we have successfully produced low amounts of the full-length PKC-ι and the catalytic domain of PKC-ι in Tuner (DE3) and BL21(DE3) bacterial cells, respectively. We have also isolated a small amount of the PKC-ι catalytic domain from BL21(DE3) using a nickel column. However, continued optimization of the bacterial environment is needed to increase the quantity and quality of our desired protein. Citation Format: Tracess Smalley, Rekha Patel, Mildred Acevedo-Duncan. Full-length bio-active atypical protein kinase c ι expression and purification in a bacterial system [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B123. doi:10.1158/1535-7163.TARG-19-B123