COMBINATORIAL PROTEIN LIBRARY SCREENING BY PERIPLASMIC EXPRESSION

摘要

The invention overcomes the deficiencies of the prior art by providing a rap id approach for isolating binding proteins capable of binding small molecules a nd peptides. In the technique, libraries of candidate binding proteins, such as antibody sequences, may be expressed in the periplasm of gram negative bacteria with at least one target ligand. In clones expressing recombinant polypeptides with affinity for the ligand, the ligand becomes bound and retained by the cell even after removal of the outer membrane, allowing the cell to be isolated from cells not expressing a binding polypeptide with affinity for the target ligand. The target ligand may be detected in numerou s ways, including use of direct fluorescence or secondary antibodies that are fluorescently labeled, allowing use of efficient screening techniques such a s fluorescence activated cell sorting (FACS). The approach is more rapid and robust than prior art methods and avoids problems associated with the outer surface~ expression of ligand fusion proteins employed with phage display.