Display cloning: functional identification of natural product receptors using cDNA-phage display

Background: The identification of cellular targets has traditionally been t he starting point for natural product mode of action studies and has led to the understanding of many biological processes. Conventional experimental approaches have depended on cell-based screening and/or affinity chromatogr aphy, Although both of these techniques aid in the discovery of protein cel lular targets, a method that couples protein identification with gene isola tion would be extremely valuable, Results: A procedure for the direct cloning of cellular proteins, based on their affinity for natural products, using cDNA phage display has been deve loped. The technique is referred to as display cloning because it involves the cloning of proteins displayed on the surface of a bacteriophage particl e. The approach has been established by isolating a full-length gene clone of FKBP12 (FK506-binding protein) from a human brain cDNA library using a b iotinylated FK506 probe molecule, During the affinity selection, the FKBP12 gene emerged as the dominant library member and was the only sequence iden tified after the second round of selection, Conclusions: The development of display cloning greatly facilitates the inv estigation of ligand-receptor interaction biology and natural product mode of action studies. This procedure utilizes heterologous protein display on infectious phage, which allows the amplification and repeated selection of putative sequences, leading to unambiguous target identification. In additi on, the direct connection of a functional protein to its gene sequence elim inates the subsequent cloning step required with tissue homogenate or cell lysate affinity methods, allowing direct isolation of an expressible gene s equence.