title: Targeting mammalian organelles with internalizing phage (iPhage) libraries.
abstract: Techniques that are largely used for protein interaction studies and the discovery of intracellular receptors, such as affinity-capture complex purification and the yeast two-hybrid system, may produce inaccurate data sets owing to protein insolubility, transient or weak protein interactions or irrelevant intracellular context. A versatile tool for overcoming these limitations, as well as for potentially creating vaccines and engineering peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage-display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries using a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and for fingerprinting functional protein domains in living cells. Here we explain the design, cloning, construction and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ∼8 weeks.
authors.affiliation: David H. Koch Center, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
authors.forename: Roberto
authors.initials: R
authors.lastname: Rangel
authors.forename: Andrey S
authors.initials: AS
authors.lastname: Dobroff
authors.forename: Liliana
authors.initials: L
authors.lastname: Guzman-Rojas
authors.forename: Carolina C
authors.initials: CC
authors.lastname: Salmeron
authors.forename: Juri G
authors.initials: JG
authors.lastname: Gelovani
authors.forename: Richard L
authors.initials: RL
authors.lastname: Sidman
authors.forename: Renata
authors.initials: R
authors.lastname: Pasqualini
authors.forename: Wadih
authors.initials: W
authors.lastname: Arap
mesh_terms: D000818:Animals; D061505:Cell Surface Display Techniques; D003001:Cloning, Molecular; D008322:Mammals; D019151:Peptide Library
chemical_list: D019151:Peptide Library
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