UsingcDNA phage display for the direct cloning of cellular proteins and functional identification of natural product receptors.

摘要

The identification of cellular targets has traditionally been the starting point for natural product mode of action studies and has subsequently led to the understanding of many biological processes. Conventional experimental approaches have depended on cell-based screening and/or affinity chromatography. Although both of these techniques aid in the discovery of protein cellular targets, a method that couples both protein identification and gene isolation would be extremely valuable.; Many techniques have been developed to better understand biological mechanisms. Among them, cDNA Phage Display combines vitro genetic expression with traditional affinity chromatography and results in a direct link between the function of a protein and its gene sequence. To elucidate natural product receptors by utilizing their affinity towards their respective binding partners, Phage Display is a procedure that uses the expression, selection, and amplification of cDNA libraries of proteins on the surface coat protein of the bacteriophage, thus a direct linkage of phenotype to genotype is achieved.; A procedure for the direct cloning of cellular proteins, based on their affinity for natural products, using cDNA phage display has been developed. The technique is referred to as Display Cloning because it involves the cloning of proteins displayed on the surface of a bacteriophage particle. 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 biotinylated FK506 probe molecule.; The development of Display Cloning greatly facilitates the investigation 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. This direct connection of a functional protein to its gene sequence eliminates the subsequent cloning step required with tissue homogenate or cell lysate affinity methods, allowing direct isolation of an expressible gene sequence.