Selection of internalizing ligand-display phage using rolling circle amplification for phage recovery.
Selection of phage libraries against complex living targets such as whole cells or organs can yield valuable targeting ligands without prior knowledge of the targeted receptor. Our previous studies have shown that noninfective multivalent ligand display phagemids internalize into mammalian cells more efficiently than their monovalent counterparts suggesting that cell-based selection of internalizing ligands might be improved using multivalently displayed peptides, antibodies or cDNAs. However, alternative methods of phage recovery are needed to select phage from noninfective libraries. To this end, we reasoned that rolling circle amplification (RCA) of phage DNA could be used to recover noninfective phage. In feasibility studies, we obtained up to 1.5 million-fold enrichment of internalizing EGF-targeted phage using RCA. When RCA was applied to a large random peptide library, eight distinct human prostate carcinoma cell-internalizing peptides were isolated within three selection rounds. These data establish RCA as an alternative to infection for phage recovery that can be used to identify peptides from noninfective phage display libraries or infective libraries under conditions where there is the potential for loss of phage infectivity.
Selective Genetics, Inc., San Diego, California 92121, USA.
Michael
M
Burg
Edward P
EP
Ravey
Michelle
M
Gonzales
Emelie
E
Amburn
Peggy Ho
PH
Faix
Andrew
A
Baird
David
D
Larocca
D001435:Bacteriophages / Q000235:genetics*; D002277:Carcinoma / Q000235:genetics; D004815:Epidermal Growth Factor / Q000302:isolation & purification / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D008024:Ligands; D008297:Male; D008967:Molecular Biology / Q000379:methods*; D019151:Peptide Library; D011471:Prostatic Neoplasms / Q000235:genetics; D014407:Tumor Cells, Cultured
D008024:Ligands; D019151:Peptide Library; D004815:Epidermal Growth Factor