Antibody-induced oligomerization and activation of an engineered reporter enzyme.
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 beta-glucuronidase (GUS). The specific activity of the purified epitope-tagged GUS variant was increased by up to approximately 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.
Geddie
Melissa L
ML
Department of Biochemistry, Center for Fundamental and Applied Molecular Evolution, Emory University School of Medicine, Rollins Research Center, Room 4119, 1510 Clifton Road, Atlanta, GA 30322, USA.
Matsumura
Ichiro
I
D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D005966:Glucuronidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D015202:Protein Engineering; D020836:Protein Structure, Quaternary
D000906:Antibodies; D005966:Glucuronidase