title: Purification of molecular machines and nanomotors using phage-derived monoclonal antibody fragments.
abstract: Molecular machines and nanomotors are sophisticated biological assemblies that convert potential energy stored either in transmembrane ion gradients or in ATP into kinetic energy. Studying these highly dynamic biological devices by X-ray crystallography is challenging, as they are difficult to produce, purify, and crystallize. Phage display technology allows us to put a handle on these molecules in the form of highly specific antibody fragments that can also stabilize conformations and allow versatile labelling for electron microscopy, immunohistochemistry, and biophysics experiments.Here, we describe a widely applicable protocol for selecting high-affinity monoclonal antibody fragments against a complex molecular machine, the A-type ATPase from T. thermophilus that allows fast and simple purification of this transmembrane rotary motor from its wild-type source. The approach can be readily extended to other integral membrane proteins and protein complexes as well as to soluble molecular machines and nanomotors.
authors.affiliation: CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Zaragoza, Spain.
authors.forename: Olga
authors.initials: O
authors.lastname: Esteban
authors.forename: Daniel
authors.initials: D
authors.lastname: Christ
authors.forename: Daniela
authors.initials: D
authors.lastname: Stock
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000915:Antibody Affinity; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D019921:Biotinylation; D061505:Cell Surface Display Techniques; D002846:Chromatography, Affinity; D004926:Escherichia coli; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000302:isolation & purification; D053758:Nanoparticles / Q000032:analysis / Q000737:chemistry; D036103:Nanotechnology; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D007128:Immunoglobulin Fragments; D020409:Molecular Motor Proteins; D006180:Proton-Translocating ATPases