Enhanced response to antibody binding in engineered β-galactosidase enzymatic sensors

Jordi X. Feliu, Neus Ferrer-Miralles, Esther Blanco, Daniel Cazorla, Francisco Sobrino, Antonio Villaverde

Research output: Contribution to journalArticleResearchpeer-review

19 Citations (Scopus)

Abstract

Peptide display on solvent-exposed surfaces of engineered enzymes allows them to respond to anti-peptide antibodies by detectable changes in their enzymatic activity, offering a new principle for biosensor development. In this work, we show that multiple peptide insertion in the vicinity of the Escherichia coli β-galactosidase active site dramatically increases the enzyme responsiveness to specific anti-peptide antibodies. The modified enzymes HD7872A and HT7278CA, carrying eight and 12 copies respectively of a foot-and-mouth disease peptide per enzyme molecule, show antibody-mediated activation factors higher than those previously observed in the first generation enzymatic sensors, for HT7278CA being close to 400%. The analysis of the signal transduction process with multiple inserted proteins strongly suggests a new, non-exclusive mechanism of enzymatic regulation in which the target proteins might be stabilised by the bound antibody, extending the enzyme half-life and consequently enhancing the signal-background ratio. In addition, the tested sensors are differently responsive to sera from immune farm animals, depending on the antigenic similarity between the B-cell epitopes in the immunising virus and those in the peptide used as sensing element on the enzyme surface. Altogether, these results point out the utility of these enzymatic biosensors for a simple diagnosis of foot-and-mouth disease in an extremely fast homogeneous assay. © 2002 Elsevier Science B.V. All rights reserved.
Original languageEnglish
Pages (from-to)212-224
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1596
DOIs
Publication statusPublished - 29 Apr 2002

Keywords

  • Antibody
  • Antigenic distance
  • Biosensor
  • Foot-and-mouth disease virus
  • Protein engineering
  • β-Galactosidase

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