Generation of anticalins with specificity for a nonsymmetric phthalic acid ester.
A set of engineered lipocalins, so-called anticalins, that bind benzyl butyl phthalate, a potential pollutant of environmental and food samples or medical plastic ware, has been generated. To this end, the synthesis of a derivative of the target analyte carrying an activatable carboxylate group at the end of an aliphatic spacer arm was established. This compound was covalently coupled to amino-functionalized paramagnetic beads. Using phage display technology three variants were selected from a random library of the bilin-binding protein (BBP), a prototypic lipocalin, which exhibit binding activity toward the nonsymmetric phthalic acid ester. These anticalins (denominated PhtA, PhtB, and PhtC) possess dissociation constants of 9.1, 6.2, and 11.6 microM, respectively. Specificity for the binding of other phthalic acid esters was studied. No cross-reactivity was found for diethyl phthalate, while binding to dibutyl phthalate was observed with higher dissociation constants. Interestingly, two differing types of binding behavior were observed among the three selected anticalins. Sequence comparison of these engineered lipocalins with the wild-type BBP revealed that all of the 16 randomized positions carried an amino acid exchange and that a certain sequence pattern had been selected, thus pointing toward a peculiar mode of structural interaction. Our data suggest that the generation of anticalins may provide an alternative to antibodies for the creation of stable receptor proteins against haptens with bioanalytical relevance.
Mercader
Josep V
JV
Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, Germany.
Skerra
Arne
A
D000818:Animals; D017151:Antibodies, Catalytic / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001709:Biotechnology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004952:Esters / Q000737:chemistry* / Q000378:metabolism; D006241:Haptens; D007301:Insect Hormones / Q000737:chemistry / Q000378:metabolism; D019476:Insect Proteins; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D010795:Phthalic Acids / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering
D017151:Antibodies, Catalytic; D002352:Carrier Proteins; D004952:Esters; D006241:Haptens; D007301:Insect Hormones; D019476:Insect Proteins; D008024:Ligands; D019151:Peptide Library; D010795:Phthalic Acids; C053849:bilin-binding protein, insect; C027561:butylbenzyl phthalate