Ligand binding of a ribosome-displayed protein detected in solution at the single molecule level by fluorescence correlation spectroscopy.
Interaction of a single-chain antibody fragment (scFv) with its cognate antigen while still attached to the ribosome was studied by fluorescence correlation spectroscopy (FCS). In experiments with purified scFv, FCS was capable of resolving the difference in diffusion time between free and antibody-bound labelled antigen. Ribosome-displayed antibody fragments generated by in vitro translation, in which neither the protein nor the mRNA leaves the ribosome owing to the absence of a stop codon and stabilizing buffer conditions, could be shown to specifically bind the antigen. The antibody-antigen interaction was specific, as shown by inhibition or displacement with unlabelled antigen and by control experiments with a non-cognate antibody fragment.
Jermutus
Lutz
L
Biochemisches Institut, Universität Zürich, Switzerland.
Kolly
Reto
R
Földes-Papp
Zeno
Z
Hanes
Jozef
J
Rigler
Rudolf
R
Plückthun
Andreas
A
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D008024:Ligands; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D036103:Nanotechnology; D025941:Protein Interaction Mapping / Q000379:methods; D012269:Ribosomal Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D008024:Ligands; D046911:Macromolecular Substances; D012269:Ribosomal Proteins