Artículos de revistas
Expression and purification of a recombinant adenovirus fiber knob in a baculovirus system
Fecha
2008Registro en:
INTERVIROLOGY, v.51, n.3, p.189-195, 2008
0300-5526
10.1159/000151532
Autor
FARINHA-ARCIERI, Luis E.
PORCHIA, Bruna M.
CARROMEU, Cassiano
SIMABUCO, Fernando M.
TAMURA, Rodrigo E.
FERREIRA, Luis C. S.
ZERBINI, Luiz F.
VENTURA, Armando M.
Institución
Resumen
Objectives: To construct a recombinant baculovirus expressing the fiber knob domain of human adenovirus type 2 modified by the insertion of a foreign peptide, purify this protein after its production in insect cells, and to test its properties. Methods: Recombinant baculoviruses expressing the fiber knob were produced in Sf9 cells. The recombinant fiber knob was recovered from culture supernatants of infected cells and purified by a combination of Ni-NTA and ion-exchange chromatography. Results: Fiber knob was recovered from the culture media as a soluble protein. In the system used, the fiber knob is expressed fused with the V5 epitope and a histidine tag, which allowed purification by Ni-NTA chromatography. The protein was further purified by ion-exchange chromatography. We show that the recombinant fiber knob produced, with 31 extra amino acids in the C-terminus, can oligomerize and bind to the adenovirus receptor CAR, as it can block the infection of a recombinant type 5 adenovirus. Conclusions: The modified form of the fiber knob, produced in insect cells and purified by Ni-NTA and ion-exchange chromatography, retains the properties of oligomerization and binding to the fiber natural receptor, CAR. This construct has the potential to be a new adjuvant. Copyright (C) 2008 S. Karger AG, Basel.