ABSTRACT
Expression of recombinant proteins with baculovirus-infected insect larvae is a scarcely investigated alternative in comparison to that in insect cell lines, a system with growing popularity in the field of biotechnology. The aim of this study was to investigate the chromatographic behavior and physicochemical properties of the proteome of Rachiplusia nu larvae infected with recombinant Autographa californica multiple nucleopolyhedrosis virus (AcMNPV), in order to design rational purification strategies for the expression of heterologous proteins in this very complex and little-known system, based on the differential absorption between target recombinant proteins and the system's contaminating ones. Two-dimensional (2D) gel electrophoresis showed differences in the protein patterns of infected and non-infected larvae. Hydrophobic interaction matrices adsorbed the bulk of larval proteins, thus suggesting that such matrices are inappropriate for this system. Only 0.03% and 2.9% of the total soluble protein from the infected larval extract was adsorbed to CM-Sepharose and SP-Sepharose matrices, respectively. Immobilized metal ion affinity chromatography represented a solid alternative because it bound only 1.4% of the total protein, but would increase the cost of the purification process. We concluded that cation-exchange chromatography is the best choice for easy purification of high-isoelectric-point proteins and proteins with arginine tags, since very few contaminating proteins co-eluted with our target protein.
Subject(s)
Histidine , Moths , Nucleopolyhedroviruses , Recombinant Fusion Proteins , Animals , Chromatography, Liquid , Histidine/biosynthesis , Histidine/chemistry , Histidine/isolation & purification , Histidine/pharmacology , Larva/chemistry , Larva/genetics , Larva/metabolism , Larva/virology , Moths/chemistry , Moths/genetics , Moths/metabolism , Moths/virology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purificationABSTRACT
Discorre-se sobre as transformaçöes bioquímicas "post-mortem" no pescado e os mecanismos de formaçäo da histamina. Evidenciam-se as bactérias responsáveis pela produçäo dessa amina e os tipos de pescado envolvidos na intoxicaçäo por ela acarretada. Discutem-se os fatores condicionantes e os meios para prevenir a intoxicaçäo histamínica