MIRANDA Maria Victoria
congresos y reuniones científicas
Physicochemical characterization of proteic contaminants from insect larvae infected with recombinant baculovirus for protein expression
MC CALLUM GREGORIO; LAUTARO BRACCO; ALEXANDRA TARGOVNIK; MARIA V MIRANDA
Santiago de Compostela
Congreso; XII EUPA Congress; 2018
Spanish Proteomics Society (SEProt) and the Portuguese Proteomics Association (ProCura),
Background: the use of baculovirus as an expression vector for the production of recombinant proteins has grown substantially in the last few years, but insect cell lines require aseptic environments, sterile equipment, bioreactors and expensive growth medium. The utilization of insect larvae constitutes an excellent alternative with high expression levels and without the need for the requirements cell lines impose, reducing costs several times. The biggest limitation for this approach is its complex purification. The aim of this work was to study the proteic contaminants present in the system, their chromatographic behavior and their physicochemical characteristics, to elaborate rational and scalable purification algorithms based on this knowledge.Methods: Rachiplusia nu larvae infected with recombinant baculovirus based on Autographa Californica, were reared for 4 days at 27°C and fed an artificial diet. Homogenates obtained were clarified and purified using different chromatographic matrixes. The eluates and total protein extract of infected and non-infected larvae were analyzed using 2D gel electrophoresis.Results: a smaller number of total proteins was detected in infected larvae compared to non-infected ones. Using cationic exchange matrixes, the number of spots detected was scarce. In contrast anion exchange matrixes adsorbed a high number of host and viral proteins.When studying hydrophobic interaction matrixes, most of the proteins detected presented pI near 7 and an average low molecular weight.To validate these results, horse radish peroxidase expressed with an 8-arginine tag to elevate its pI from 7.0 to 9.5 was purified using CM-sepharose and SP-sepharose matrixes, obtaining high levels of purity with the latter.Conclusion: in conclusion, the study of chromatographic behavior and physicochemical characterization of the system?s contaminants, allowed us to design and optimize rational purification processes. The predominance of acidic proteins represents an opportunity to easily purify proteins with high isoelectric points using CM and SP-sepharose chromatography.