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Generation of Conformation-Specific Antibody Fragments for Crystallization of the Multidrug Resistance Transporter MdfA

  • Frank Jaenecke
  • Yoshiko Nakada-Nakura
  • Kumar Nagarathinam
  • Satoshi Ogasawara
  • Kehong Liu
  • Yunhon Hotta
  • So Iwata
  • Norimichi Nomura
  • Mikio Tanabe
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1700)

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.

Key words

Multidrug resistance transporter Crystallization Antibody fragment MFS transporter Stabilization 

Notes

Acknowledgements

We thank Dr. Alexander Cameron for critically reading the manuscript. The work was supported by the Bundesministerium für Bildung und Forschung (BMBF) ZIK program (FKZ 03Z2HN21), by the ERDF (1241090001) (M.T.), by the ERATO Human Receptor Crystallography Project of the Japan Science and Technology Agency (JST) (S.I.), by the Research Acceleration Program of the JST (S.I.), by the Targeted Proteins Research Program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (S.I.), and by Grants-in-Aids for Scientific Research from the MEXT (No. 22570114 to N.N.). The crystallographic data were tested at Swiss light source (SLS, Villingen) with supported by the funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under BioStruct-X (grant agreement N°283570, project ID: BioStructx_5450). The authors declare no conflict of interest. Frank Jaenecke and Yoshiko Nakada-Nakura have contributed equally to this work.

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Frank Jaenecke
    • 1
  • Yoshiko Nakada-Nakura
    • 2
    • 3
  • Kumar Nagarathinam
    • 1
  • Satoshi Ogasawara
    • 2
    • 4
  • Kehong Liu
    • 2
  • Yunhon Hotta
    • 2
  • So Iwata
    • 2
    • 3
    • 5
    • 6
  • Norimichi Nomura
    • 2
    • 3
    • 5
  • Mikio Tanabe
    • 1
    • 7
  1. 1.HALOmem, Membrane Protein BiochemistryMartin-Luther-University Halle-WittenbergHalle (Saale)Germany
  2. 2.Department of Cell Biology, Graduate School of MedicineKyoto UniversityKyotoJapan
  3. 3.Research Acceleration Program, Membrane Protein Crystallography ProjectJapan Science and Technology AgencySakyo-kuJapan
  4. 4.Department of Chemistry, Graduate School of ScienceChiba UniversityChibaJapan
  5. 5.ERATO, Iwata Human Receptor Crystallography ProjectJapan Science and Technology AgencyKyotoJapan
  6. 6.RIKEN, SPring-8 CenterSayoJapan
  7. 7.Structural Biology Research Center, Photon Factory, Institute of Materials Structure ScienceHigh Energy Accelerator Research Organization (KEK)IbarakiJapan

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