Nucleic Acids Research Advance Access originally published online on October 26, 2007
Nucleic Acids Research 2007 35(20):e138; doi:10.1093/nar/gkm914
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Nucleic Acids Research, 2007, Vol. 35, No. 20 e138
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
A novel method for generating and screening peptides and libraries displayed on adenovirus fiber
James Buchanan Brady Urology Institute, Johns Hopkins University School of Medicine, Broadway Research Building 467, 733N Broadway, Baltimore, MD 21205, USA
*To whom correspondence should be addressed. Tel: 410-502-4822; Fax: 410-502-1853; Email: slupold{at}jhmi.edu
Received July 30, 2007. Revised September 25, 2007. Accepted October 8, 2007.
Capsid-displayed adenoviral peptide libraries have been a significant, yet unfeasible goal in biotechnology. Three barriers have made this difficult: the large size of the viral genome, the low efficiency of converting plasmid-based genomes into packaged adenovirus and the fact that library amplification is hampered by the ability of two (or more) virus to co-infect one cell. Here, we present a novel vector system, pFex, which is capable of overcoming all three barriers. With pFex, modified fiber genes are recombined into the natural genetic locus of adenovirus through unidirectional Cre–lox recombination. Modified-fiber genes can be directly shuttled into replicating viral genomes in mammalian cells. The ‘acceptor’ vector does not contain the fiber gene, and therefore does not propagate until it has received a ‘donor’ fiber gene. Therefore, This methodology overcomes the low efficiency of transfecting large viral genomes and bypasses the need for transition to functional virus. Thus, with a fiber-shuttle library, one can generate and evaluate large numbers of fiber-modified adenovirus simultaneously. Finally, successful fiber genes can be rescued from virus and recombined back into shuttle plasmids, avoiding the need to propagate mixed viral pools. For proof of principal, we use this new system to screen a capsid-displayed peptide library for retargeted viral infection.