RESUMO
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.
Assuntos
Histidina , Mariposas , Nucleopoliedrovírus , Proteínas Recombinantes de Fusão , Animais , Cromatografia Líquida , Histidina/biossíntese , Histidina/química , Histidina/isolamento & purificação , Histidina/farmacologia , Larva/química , Larva/genética , Larva/metabolismo , Larva/virologia , Mariposas/química , Mariposas/genética , Mariposas/metabolismo , Mariposas/virologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Due to the importance of fever and its correct measurement, we try to describe in our study the use of the thermometer by a sample of mothers of young children chosen at random in a low-middle class neighborhood. We surveyed them at home and checked their use of the thermometer. We found a frequent and correct usage of the thermometer. We identify the mother's educational level as one indicator of some of the mistakes in their use of the thermometer. We propose specific points to teach every mother when she goes to the pediatrician in order to provide adequate use of the thermometer in children with fever.