Targeting mammalian organelles with internalizing phage (iPhage) libraries

Roberto Rangel, Andrey S. Dobroff, Liliana Guzman-Rojas, Carolina C. Salmeron, Juri G. Gelovani, Richard L. Sidman, Renata Pasqualini, Wadih Arap

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1916-1939
Number of pages24
JournalNature Protocols
Volume8
Issue number10
DOIs
StatePublished - Oct 2013
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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