Recombinant peroxidase production in species of Lepidoptera

ROMERO L.; TARGOVNIK A; FOGAR M.; SIMONELLA M.; CASCONE O; MIRANDA MV

Barcelona

Congreso; 14 th European Congress on Biotechnology.; 2009

Baculovirus-insect cell system is a popular choice for heterologous gene expression when an eukaryotic environment is required. Autographa californica >multiple nucleopolyhedrovirus (AcMNPV) is the most studied baculovirus expression vector. Its relatively wide host range of Lepidoptera-Noctuidae larvae compared to other baculoviruses makes it a powerful tool for recombinant protein expression in these “bio-factories”. In vivo expression system seems to be economically more advantageous than insect cell culture due to its lower cost and higher protein yield. In Argentina, many important crops such as soy and corn are frequently affected by insect plagues including Rachiplusia nu, Spodoptera frugiperda and Helicoverpa zea. Horseradish peroxidase isozyme C (HRPC) is an important commercial biocatalyst. Its complex structure complicates its recombinant expression in prokaryotes in active form. Two recombinant viruses were constructed to compare the performance of HRPC expression in the aforementioned Lepidopteran hosts. The first (AcMNPVHRPC occ-) was formed by introducing the HRPC gene in locus polyhedrin so it resulted in an intrahaemocoelically infective virus. The second (AcMNPVHRPC occ+) was identical except for polyhedrin gene presence under p10 promoter to achieve polyhedra production capability for oral administration. Each species was infected with both viruses and harvesting day post-infection (dpi) was optimized. All species were successfully infected by AcMNPVHRPC occ-: S. frugiperda (7dpi), R. nu (4 dpi) and H. zea (6 dpi) expressed 312, 122 and 38 mg HRPC/kg larva, respectively. On the other hand, only R. nu was highly susceptible to oral infection. Injection of both viruses produced practically the same expression kinetic profile; therefore, polyhedrin gene presence under p10 promotor does not affect HRPC expression. According to these results, AcMNPVHRPC occ+ could be used intrahaemocoelically with the great advantage of being also suitable for oral infection of permissive Lepidoptera. Oral administration reduces larvae death risk because of manipulation and is easier to scale-up. However, AcMNPVHRPC occ- infection efficiency was 100% in all experiments and HRPC levels were higher compared with AcMNPVHRP occ+ infection. In conclusion, the design of the most suitable and profitable strategy will depend on the host availability and the protein amount required.