Chapter title |
Generation of Conformation-Specific Antibody Fragments for Crystallization of the Multidrug Resistance Transporter MdfA
|
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Chapter number | 7 |
Book title |
Bacterial Multidrug Exporters
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7454-2_7 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7452-8, 978-1-4939-7454-2
|
Authors |
Frank Jaenecke, Yoshiko Nakada-Nakura, Kumar Nagarathinam, Satoshi Ogasawara, Kehong Liu, Yunhon Hotta, So Iwata, Norimichi Nomura, Mikio Tanabe |
Abstract |
A major hurdle in membrane protein crystallography is generating crystals diffracting sufficiently for structure determination. This is often attributed not only to the difficulty of obtaining functionally active protein in mg amounts but also to the intrinsic flexibility of its multiple conformations. The cocrystallization of membrane proteins with antibody fragments has been reported as an effective approach to improve the diffraction quality of membrane protein crystals by limiting the intrinsic flexibility. Isolating suitable antibody fragments recognizing a single conformation of a native membrane protein is not a straightforward task. However, by a systematic screening approach, the time to obtain suitable antibody fragments and consequently the chance of obtaining diffracting crystals can be reduced. In this chapter, we describe a protocol for the generation of Fab fragments recognizing the native conformation of a major facilitator superfamily (MFS)-type MDR transporter MdfA from Escherichia coli. We confirmed that the use of Fab fragments was efficient for stabilization of MdfA and improvement of its crystallization properties. |
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