Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold

We demonstrate that the ligand pocket of a lipocalin from Pieris brassicae, the bilin-binding protein (BBP), can be reshaped by combinatorial protein design such that it recognizes fluorescein, an established immunological ha pten. For this purpose 16 residues at the center of the binding site, which is formed by four loops on top of an eight-stranded beta-barrel, were subj ected to random mutagenesis. Fluorescein-binding BBP variants were then sel ected from the mutant Library by bacterial phage display, Three variants we re identified that complex fluorescein with high affinity, exhibiting disso ciation constants as low as 35.2 nM. Notably, one of these variants effects almost complete quenching of the ligand fluorescence, similarly as an anti -fluorescein antibody. Detailed ligand-binding studies and site directed mu tagenesis experiments indicated (i) that the molecular recognition of fluor escein is specific and (ii) that charged residues at the center of the pock et are responsible for tight complex formation. Sequence comparison of the BBP variants directed against fluorescein with the wild-type protein and wi th further variants that were selected against several other ligands reveal ed that all of the randomized amino acid positions are variable. Hence, a l ipocalin can be used for generating molecular pockets with a diversity of s hapes. We term this class of engineered proteins "anticalins". Their one-do main scaffold makes them a promising alternative to antibodies to create a stable receptor protein for a ligand of choice.