title: A method for measuring binding constants using unpurified in vivo biotinylated ligands.
abstract: Obtaining accurate kinetics and steady-state binding constants for biomolecular interactions normally requires pure and homogeneous protein preparations. Furthermore, in many cases, one of the ligands must be labeled. Over the past decade, several technologies have been introduced that allow for the measurement of kinetics constants for multiple different interactions in parallel. One such technology is bio-layer interferometry (BLI), which has been used to develop systems that can measure up to 96 biomolecular interactions simultaneously. However, despite the ever-increasing throughput of the tools available for measuring protein-protein interactions, the preparation of pure protein still remains a bottleneck in the process of producing high-quality kinetics data. Here, we show that high-quality binding data can be obtained using soluble lysate fractions containing protein that has been biotinylated in vivo using BirA and then applied to BLI sensors without further purification. Furthermore, we show that BirA ligase does not necessarily need to be co-overexpressed with the protein of interest for biotinylation of the biotin acceptor peptide to occur, suggesting that the activity of endogenous BirA in Escherichia coli is sufficient for producing enough biotinylated protein for a binding experiment.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Anastassia K
authors.initials: AK
authors.lastname: Pogoutse
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Christine Chieh-Lin
authors.initials: CC
authors.lastname: Lai
authors.affiliation: Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Ostan
authors.affiliation: Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Rong-hua
authors.initials: RH
authors.lastname: Yu
authors.affiliation: Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Anthony B
authors.initials: AB
authors.lastname: Schryvers
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Electronic address: trevor.moraes@utoronto.ca.
authors.forename: Trevor F
authors.initials: TF
authors.lastname: Moraes
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006801:Humans; D007368:Interferometry / Q000379:methods*; D008024:Ligands; D008025:Ligases / Q000378:metabolism; D009345:Neisseria meningitidis / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D012097:Repressor Proteins / Q000378:metabolism*; D014168:Transferrin / Q000378:metabolism; D033882:Transferrin-Binding Protein B / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D012097:Repressor Proteins; D014168:Transferrin; D033882:Transferrin-Binding Protein B; C471607:UBE2N protein, human; C114715:UBE2V2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli
keywords: Bio-layer interferometry (BLI); Biomolecular interactions; BirA; Dissociation constant (K(D)); In vivo biotinylation; Lysate