Antibody-Induced Oligomerization and Activation of an Engineered Reporter Enzyme

• Published in: Journal of molecular biology Vol. 369; no. 4; pp. 1052 - 1059
• Main Authors: Geddie, Melissa L., Matsumura, Ichiro
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 15.06.2007
• Subjects: Antibodies - chemistry; Antibodies - metabolism; biosensor; Enzyme Activation; Glucuronidase - chemistry; Glucuronidase - genetics; Glucuronidase - metabolism; high-throughput screen; Humans; induced dimerization; Models, Molecular; molecular switch; Mutagenesis; Protein Engineering; Protein Structure, Quaternary; reporter enzyme; anti-HA; high-throughput screen; pNP; biosensor; reporter enzyme; GUS; molecular switch; GST; pNP-gluc; induced dimerization; X-gluc; MUG
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2007.03.076

Our objective is to produce a protein biosensor (or molecular switch) that is specifically activated in solution by a monoclonal antibody. Many effector-dependent enzymes have evolved in nature, but the introduction of a novel regulatory mechanism into a normally unregulated enzyme poses a difficult design problem. We used site-saturation mutagenesis and screening to generate effector-activated variants of the reporter enzyme β-glucuronidase (GUS). The specific activity of the purified epitope-tagged GUS variant was increased by up to ∼500-fold by the addition of an equimolar concentration of a monoclonal antibody. This molecular switch is modular in design, so it can easily be re-engineered for the detection of other peptide-specific antibodies. Such antibody-activated reporters could someday enable point-of-care serological assays for the rapid detection of infectious diseases.