Expression of Canine Kynurenine 3-Monooxygenase by Baculovirus for Canine Mammary Tumor Diagnosis

Abstract

Received: Revised: Accepted: Published online: July 28, 2019 January 05, 2020 January 09, 2020 February 03, 2020 Kynurenine 3-monooxygenase (KMO), an outer mitochondrial membrane enzyme that is central to the kynurenine pathway, has been demonstrated to be associated with malignancy in human cancers. Cancers in dogs are considered reliable and clinically relevant models of human diseases owing to similarities in the natural history of these human and canine tumors and the superficial resemblances in the anatomy, topology, metastatic patterns, and response to therapy between canine and human cancers. This study aims to establish an efficient protocol to prepare the recombinant canine KMO protein for potential application in canine cancer study. The amino acid sequence and structure of canine KMO were analyzed using homology modeling provided by SWISS-MODEL. The canine KMO (cKMO) was produced by using a baculovirus–insect cell (Sf9 cells) expression system. Fulllength cKMO was expressed by the baculovirus-infected Sf9 cells as a 477-aminoacid protein with a molecular weight of 55 kDa. On average a yield of 2 mg of protein was obtained from 2 × 10 baculovirus-infected Sf9 cells. The results from western blot and immunofluorescent assay showed KMO can be successfully expressed by Sf9 cells within the cytosol and mitochondria. The purified recombinant KMO protein could be used as an antigen for generating anti-cKMO antibodies to further investigate the role in canine carcinogenesis. ©2020 PVJ. All rights reserved