Preparation of baculovirus transfer vectors for in vitro production of the P4501A1 and P4503A4 isoenzymes

: The aim of this work is the construction of an expression system for in vitro synthesis of microsomal monooxygenases P4501A1 and P4503A4, which catalyze oxidative transformations of most xenobiotics in both animal and human organisms. cDNAs encoding both proteins were obtained following the UBMTA protocol by the courtesy of holders, and amplified by established methods. Baculovirus transfer vectors were used to clone these cDNAs. These vectors contain a strong polyhedrine promoter surrounded by sequences homologous to that of baculovirus DNA, allowing the recombination of the vector with the viral DNA, and hence the production of a protein. Established methods and PCR were used to insert cDNA into the vectors, and the insertion was verified by the PCR method with specific primers and using restriction endonucleases.