TY - JOUR
T1 - Targeting mammalian organelles with internalizing phage (iPhage) libraries
AU - Rangel, Roberto
AU - Dobroff, Andrey S.
AU - Guzman-Rojas, Liliana
AU - Salmeron, Carolina C.
AU - Gelovani, Juri G.
AU - Sidman, Richard L.
AU - Pasqualini, Renata
AU - Arap, Wadih
N1 - Funding Information:
acknoWleDGMents This work was supported by grants from the US National Institutes of Health, the US Department of Defense, and by awards from The University of Texas M.D. Anderson Cancer Center Trust, the Marcus Foundation, AngelWorks and the Gillson-Longenbaugh Foundation (all to W.A. and R.P.). R.R. received support from the Odyssey Scholar Program at the University of Texas M.D. Anderson Cancer Center.
PY - 2013/10
Y1 - 2013/10
N2 - 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.
AB - 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.
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U2 - 10.1038/nprot.2013.119
DO - 10.1038/nprot.2013.119
M3 - Article
C2 - 24030441
AN - SCOPUS:84884996236
SN - 1754-2189
VL - 8
SP - 1916
EP - 1939
JO - Nature protocols
JF - Nature protocols
IS - 10
ER -