title: Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector.
abstract: While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors.
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA.
authors.forename: Samuel K
authors.initials: SK
authors.lastname: Campos
authors.forename: M Brandon
authors.initials: MB
authors.lastname: Parrott
authors.forename: Michael A
authors.initials: MA
authors.lastname: Barry
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001360:Avidin / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D019921:Biotinylation / Q000379:methods*; D002213:Capsid / Q000737:chemistry / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D036022:Capsid Proteins; D010455:Peptides; C018315:hexon capsid protein, Adenovirus; D001360:Avidin; D001710:Biotin