info:eu-repo/semantics/article
Recombinant protein purification in baculovirus-infected Rachiplusia nu larvae: An approach towards a rational design of downstream processing strategies based on chromatographic behavior of proteins
Fecha
2019-02Registro en:
Mc Callum, Gregorio Juan; Arregui, Mariana Bernadett; Smith, Ignacio; Bracco, Lautaro Fidel; Wolman, Federico Javier; et al.; Recombinant protein purification in baculovirus-infected Rachiplusia nu larvae: An approach towards a rational design of downstream processing strategies based on chromatographic behavior of proteins; Academic Press Inc Elsevier Science; Protein Expression and Purification; 158; 2-2019; 44-50
1046-5928
CONICET Digital
CONICET
Autor
Mc Callum, Gregorio Juan
Arregui, Mariana Bernadett
Smith, Ignacio
Bracco, Lautaro Fidel
Wolman, Federico Javier
Cascone, Osvaldo
Targovnik, Alexandra Marisa
Miranda, Maria Victoria
Resumen
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 cationexchange 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.