High-Resolution Crystallographic Analysis of AcrB Using Designed Ankyrin Repeat Proteins (DARPins)
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X-ray crystallography is still the most prominent technique in use to decipher the 3D structures of membrane proteins. For successful crystallization, sample quality is the most important parameter that should be addressed. In almost every case, highly pure, monodisperse, and stable protein sample is a prerequisite. Vapor diffusion is in general the method of choice for obtaining crystals. Here, we discuss a detailed protocol for overproduction and purification of the inner-membrane multidrug transporter AcrB and of DARPins, which are used for crystallization of the AcrB/DARPin complex, resulting in high-resolution diffraction and subsequent structure determination.
Key wordsAcrB Resistance Nodulation cell Division Multidrug resistance Antibiotic resistance DARPin Protein crystallography
Work in the Pos lab is supported by the German Research Foundation (SFB 807, Transport and Communication across Biological Membranes and FOR2251, Adaptation and persistence of the emerging pathogen Acinetobacter baumannii), the DFG-EXC115 (Cluster of Excellence Macromolecular Complexes at the Goethe-University Frankfurt), Innovative Medicines Initiative Joint Undertaking Project Translocation (IMI-Translocation), EU Marie Curie Actions ITN, Human Frontiers Science Program (HFSP) and the German-Israeli Foundation (GIF).
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