The Xfoo expression vector based on lambda phage has been developed to produce foreign proteins fused to the phage coat proteins on the surface of the phage particle. This vector is suited for the construction of peptide libraries and cDNA libraries, since foreign proteins are produced as fusions at the carboxyl terminus of the phage coat proteins. In principle, the libraries can be searched for in vitro by affinity chromatography or by "panning" with a microtiter plate. As an application of this phage expression system, we have devised a new method for epitope mapping for monoclonal antibodies (MoAbs). Epitopes for MoAbs against human galectin-3 have been determined by the following methods. Plasmid DNA, -4.8 kb in size, containing the human galectin-3 cDNA was randomly sheared by DNase I into 50-120 bp DNA in size, ligated with oligonucleotide adaptors, and cloned into the Xfoo vector DNA. This phage library was selected by MoAbs against galectin-3 that were coated on plastic wells, and the recovered phage plaques were stained with the MoAbs. One cycle of this screening was sufficient enough to identify more than ten positive phages by a W-fold enrichment factor. Amino acid sequences of inserts from the recovered phage clones were determined by DNA sequencing and aligned to define the region that was recognized by the MoAbs. After sequencing five positive clones , regions consisting of 15 and 21 amino acid residues were determined for two distinct MoAbs, respectively. Thus, the Xfoo system can be an economical and efficient alternative to the conventional epitope mapping.
|Original language||English (US)|
|State||Published - 1996|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology