Protocol - Recombinant expression of human COX2 in insect cells using the Bac-to-Bac system and purification by ion exchange and size-exclusion chromatography

This protocol describes the recombinant expression of human cyclooxygenase-2 (COX2) encoded by the PTGS2 gene (RefSeq NM_000963.4) in Spodoptera frugiperda (Sf9) insect cells using the Bac-to-Bac baculovirus expression system. The method includes transformation of E. coli DH10Bac with pFastBac1 vector containing the full-length human PTGS2 gene, supplied pre-assembled by GenScript, followed by selection and screening of recombinant colonies, purification and PCR verification of the bacmid DNA, transfection of Sf9 cells, and production of P1–P3 viral stocks. Following pilot expression assays, recombinant COX2 protein is purified from infected insect cells using a detergent-based lysis protocol followed by ion exchange and size-exclusion chromatography, adapted from Gierse et al. (Cyclooxygenases: Methods and Protocols, 2016, Chapter 3). This strategy enables recovery of active, untagged COX2 protein suitable for biochemical and celullar studies with the COX2 protein.