title: Reporter Immobilization Assay (REIA) for Bioconjugating Reactions.
abstract: Enzymes able to ligate biomolecules are emerging tools to generate site-specific bioconjugates. In this study we present a detection and screening method for bioconjugating enzymes which overcomes limitations of analytical methods such as HPLC or MS. These techniques are experimentally demanding and often limited in sensitivity and throughput compared to enzymatic assays. The principle of this Reporter Immobilization Assay (REIA) is the ligation of a reporter enzyme to a peptide carrying an affinity handle, which can be utilized for its isolation. The REIA system exhibits a high sensitivity with a linear range down to 1 μg/mL (55 nM), a variation coefficient of 6.5%, and can be performed cost-efficiently in 96-well microtiter plate format. The application of this assay allowed the characterization of a thiol transpeptidase sortase from S. aureus which is an important drug target and a biotechnological tool for ligation and modification of proteins. Thereby, yet-undetectable promiscuous activity of sortase could be detected, e.g., the acceptance of alanine as nucleophile. In addition, we were able to provide evidence that the REIA is suitable for high throughput screening of enzyme libraries using crude cellular extract with a throughput of 600 samples per hour.
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University , 52074 Aachen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Schatte
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University , 52074 Aachen, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Bocola
authors.forename: Teresa
authors.initials: T
authors.lastname: Roth
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University , 52074 Aachen, Germany.
authors.forename: Ronny
authors.initials: R
authors.lastname: Martinez
authors.forename: Erhard
authors.initials: E
authors.lastname: Kopetzki
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University , 52074 Aachen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
authors.forename: Mara
authors.initials: M
authors.lastname: Bönitz-Dulat
mesh_terms: D000595:Amino Acid Sequence; D057075:Enzyme Assays / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010458:Peptidyl Transferases / Q000737:chemistry / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology
chemical_list: D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; D010455:Peptides; D042843:Glucose 1-Dehydrogenase; D010458:Peptidyl Transferases