Screening of Peptide Ligands for Pyrroloquinoline Quinone Glucose Dehydrogenase Using Antagonistic Template-Based Biopanning

Abstract

We have developed a novel method, antagonistic template-based biopanning, for screening peptide ligands specifically recognizing local tertiary protein structures. We chose water-soluble pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH-B) as a model enzyme for this screening. Two GDH-B mutants were constructed as antagonistic templates; these have some point mutations to induce disruption of local tertiary structures within the loop regions that are located at near glucose-binding pocket. Using phage display, we selected 12-mer peptides that specifically bound to wild-type GDH-B but not to the antagonistic templates. Consequently, a peptide ligand showing inhibitory activity against GDH-B was obtained. These results demonstrate that the antagonistic template-based biopanning is useful for screening peptide ligands recognizing the specific local tertiary structure of proteins.

Engineering PQQ-glucose dehydrogenase into an allosteric electrochemical Ca2+ sensor

“…We then used an established colorimetric assay to analyse the activity of the GDH-CaM chimeric protein. 17 As can be seen in Fig. 2, in the absence of Ca 2+ ions, the GDH-CaM displayed virtually no enzymatic activity.…”