by Keyword: X-ray crystallography
Esteban, O., Christ, D., Stock, D., (2013). Purification of molecular machines and nanomotors using phage-derived monoclonal antibody fragments Protein Nanotechnology - Methods in Molecular Biology (ed. Gerrard, J. A.), Humana Press (New York, USA) 996, 203-217
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.
JTD Keywords: ATP synthase, Crystallization, Domain antibodies, Electron microscopy, Labelling, Membrane proteins, Monoclonal antibody fragments, Phage display, Protein purification, X-ray crystallography