Phage Display Shot-Gun Cloning of Ligand-Binding Domains of Prokaryotic Receptors Approaches 100% Correct Clones

Abstract

We recently presented an application of the phage display technique enabling cloning of DNA encoding ligand-binding domain(s) of prokaryotic receptors directly from chromosomal DNA. Here we show that the use of a gene VIII-based, instead of a gene III-based, phagemid vector system results in a much more efficient selection for phage displaying a binding capacity. A phagemid library was made by insertion of randomly fragmented chromosomal DNA from Staphylococcus aureus strain 8325-4 into gene VIII in the constructed phagemid vector pG8H6. The library, which in theory should express parts of all proteins encoded by the bacterial genome, was affinity panned against the ligands IgG, fibronectin and fibrinogen, respectively. After a second panning against the same ligand, a significant increase in the number of eluted phagemid particles was observed, and 75%–100% of randomly picked clones contained inserts derived from genes encoding proteins with a binding affinity for the respective ligand. The results show that this technique can be used for cloning prokaryotic receptor genes without any prior knowledge of the receptor, thus eliminating the need for probes in the identification of receptor genes.