Expression and purification of a recombinant adenovirus fiber knob in a baculovirus system.
OBJECTIVE
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.
Farinha-Arcieri
Luis E
LE
Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brasil.
Porchia
Bruna M
BM
Carromeu
Cassiano
C
Simabuco
Fernando M
FM
Tamura
Rodrigo E
RE
Ferreira
Luis C S
LC
Zerbini
Luiz F
LF
Ventura
Armando M
AM
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D005822:Genetic Vectors; D011485:Protein Binding; D011991:Receptors, Virus / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D051603:Recoverin / Q000378:metabolism; D018411:Spodoptera
D036022:Capsid Proteins; C496731:RCVRN protein, human; D011991:Receptors, Virus; D011994:Recombinant Proteins; C018315:hexon capsid protein, Adenovirus; D051603:Recoverin