Intracellular antibodies for proteomics.
The intracellular antibody technology has many applications for proteomics studies. The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection of antibodies readily folded for ectopic expression. IACT has been used for the rapid and effective identification of novel antigen-antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by selected intracellular antibodies. IACT opens the way to the use of intracellular antibody technology for large-scale applications in proteomics. In its present format, its use is however somewhat limited by the need of a preselection of the input phage antibody libraries on protein antigens or by the construction of an antibody library from mice immunized against the target protein(s), to provide an enriched input library to compensate for the suboptimal efficiency of transformation of the yeast cells. These enrichment steps require expressing the corresponding proteins, which represents a severe bottleneck for the scaling up of the technology. We describe here the construction of a single pot library of intracellular antibodies (SPLINT), a naïve library of scFv fragments expressed directly in the yeast cytoplasm in a format such that antigen-specific intrabodies can be isolated directly from gene sequences, with no manipulation whatsoever of the corresponding proteins. We describe also the isolation from SPLINT of a panel of intrabodies against a number of different proteins. The application of SPLINT on a genome-wide scale should help the systematic study of the functional organization of cell proteome.
Visintin
Michela
M
Lay Line Genomics SpA, Via di Castel Romano 100, 00128 Rome, Italy.
Meli
Giovanni Antonio
GA
Cannistraci
Isabella
I
Cattaneo
Antonino
A
D005822:Genetic Vectors; D023281:Genomics / Q000379:methods; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D019151:Peptide Library; D010957:Plasmids; D040901:Proteomics / Q000379:methods*
D007135:Immunoglobulin Variable Region; D019151:Peptide Library