抄録
To demonstrate the practical use of a novel high-throughput screening system by single cells constructed by the molecular display method, a yeast cell chip microchamber array was developed. As applications, peptides, peptidases, and antibodies were examined. Neurolysin originally recognizes substrates with six-amino-acid-long residues, cleaving a peptide bond in the center position of the substrate amino acid sequence. To alter the recognition of the P2' amino acid of substrates by neurolysin, six residues of neurolysin which might be involved in the formation of the neurolysin S2' subsite were individually and comprehensively substituted by semirational mutagenesis coupled with the yeast molecular display system. The protein libraries of mutant neurolysins were displayed on the yeast cell surface and screening was carried out using two fluorescence-quenching peptides, the matrix metalloproteinase-2/9- and MMP-3-specific substrates. Among mutant neurolysins, one mutant neurolysin with a marked change in substrate specificity was successfully obtained. Furthermore, skillful display of antibodies (H and L chains) on the cell surface of yeast cells suggested the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach. Accordingly, the combination of the molecular display and combinatorial bioengineering would lead to produce novel medicines.
