Recombinant Protease Production in Drosophila

To produce the near full-length of CLIPB9 and CLIPA14 transcripts for eukaryotic expression, cDNA was amplified by PCR using specific primers (Table S1). The PCR product was cloned into the pMT-BiP/V5-HisA vector (Invitrogen). Putative cleavage sites of proCLIPB9 and proCLIPA14 are IGMR¹³⁶ and LGFR¹⁶³. The four residues of the hypothetical cleavage site were replaced with the IEGR tetrapeptide recognized by Factor Xa (New England Biolabs) by the overlap extension PCR method, and the recombinant plasmids of proCLIPB9_Xa and proCLIPA14_Xa were obtained. The recombinant plasmid and pCoHygro hygromycin selection vector (Invitrogen) were co-transfected into Drosophila S2 cells cultured in SFX medium (HyClone) at 28 °C, and stable cell lines were selected. After induction with 500 μM copper sulfate, the supernatants were collected for recombinant protein purification. Then, protein was purified using Ni-NTA agarose. The concentration of the purified protein was estimated by bicinchoninic acid (BCA) assay and analyzed using SDS-PAGE followed by immunoblotting. As for recombinant PPO3, it was purified with Ni-NTA agarose as described previously (31 (link)). The purified products were stored at −80 °C until use.