Manipulating adenoviral vector ion-exchange chromatography: Hexon versus fiber.
The serotype specificity of adenovirus ion-exchange chromatography has previously been studied using standard particle-based columns, and the hexon protein has been reported to determine retention time. In this study, we have submitted Adenovirus type 5 recombinants to anion-exchange chromatography using methacrylate monolithic supports. Our experiments with hexon-modified adenoviral vectors show more precisely that the retention time is affected by the substitution of amino acids in hypervariable region 5, which lies within the hexon DE1 loop. By exploring the recombinants modified in the fiber protein, we have proven the previously predicted chromatographic potential of this surface constituent. Modifications that preserve the net charge of the hexon protein, or those that cause only a small charge difference in the fiber protein, in addition to shortening the fiber shaft, did not change the chromatographic behavior of the adenovirus particles. However, modifications that include the deletion of just two negatively charged amino acids in the hexon protein, or the introduction of a heterologous fiber protein, derived from another serotype, revealed recognizable changes in anion-exchange chromatography. This could be useful in facilitating chromatography-approach purification by creating targeted capsid modifications, thereby shifting adenovirus particles away from particular interfering substances present in the crude lysate.
Department of Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia.
Jelena
J
Ruščić
Division of Molecular Biology, Laboratory for Cell Biology and Signaling, Ruđer Bošković Institute, Zagreb, Croatia.
Andreja
A
Ambriović-Ristov
Division of Molecular Biology, Laboratory for Cell Biology and Signaling, Ruđer Bošković Institute, Zagreb, Croatia.
Dragomira
D
Majhen
Department of Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia.
Sandra
S
Kolundžija
BIA Separations, Ajdovščina, Slovenia.
Miloš
M
Barut
Univ Paris-Sud, Orsay Cedex, France.|CNRS UMR 8203, Vectorologie et thérapeutiques anticancéreuses, Gustave Roussy, Villejuif Cedex, France.
Karim
K
Benihoud
Department of Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia.
Mladen
M
Krajačić
D000256:Adenoviridae; D019943:Amino Acid Substitution; D036022:Capsid Proteins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005822:Genetic Vectors
D036022:Capsid Proteins; C018315:hexon capsid protein, Adenovirus
Anion-exchange chromatography; Fiber protein; Hexon protein; Monolithic columns; Recombinant adenoviral vectors