Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector

Samuel K Campos, M. Brandon Parrott, Michael A. Barry

Research output: Contribution to journalArticle

71 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)942-954
Number of pages13
JournalMolecular Therapy
Volume9
Issue number6
DOIs
StatePublished - Jun 2004
Externally publishedYes

Fingerprint

Avidin
Biotin
Ligands
Proteins
Peptides
Adenoviridae
Virion
Biotinylation
Transferrin Receptors
Tropism
Capsid Proteins
Viral Proteins
Protein Transport
Transferrin
Anti-Idiotypic Antibodies
Viruses

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector. / Campos, Samuel K; Parrott, M. Brandon; Barry, Michael A.

In: Molecular Therapy, Vol. 9, No. 6, 06.2004, p. 942-954.

Research output: Contribution to journalArticle

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