Screening an Expression Library with a DNA Binding Site

The molecular cloning of a sequence-specific DNA binding protein using the recognition binding site as a probe was first described in 1988 (1 ). An Epstein-Barr virus protein (EBNA 1) and its DNA binding site (oriP) were instrumental in the development of this cloning technique (2 ). An oriP probe containing two high-affinity binding sites for EBNA1 was used to probe λEB, a λgt11 recombinant encoding a βgalactosidase fusion protein containing the DNA binding domain of EBNA 1 (1 ). A similar pattern of plaques on nitrocellulose replica filters was recognized with the oriP probe and with anti-EBNA1 antibody probes confirming the specificity and sensitivity of the “binding site” screening procedure. The technique was subsequently used to isolate a cDNA encoding a protein with binding affinity for the major histocompatibility complex (MHC) class I enhancer (1 ).