Skip to main content
SpringerLink
Log in

Generation of Synthetic Antibody Fragments to Detergent Solubilized Membrane Proteins

  • Protocol
  • First Online:
Book cover Chemical and Synthetic Approaches in Membrane Biology

Part of the book series: Springer Protocols Handbooks ((SPH))

  • 637 Accesses

Abstract

Structural determination of membrane proteins is extremely challenging due to the physical characteristics of membrane proteins themselves and the lack of adequate tools and technologies to perform the studies. Recent developments in micro-focus X-ray beams, novel detergents, protein thermo-stabilization, and protein engineering have been essential in expanding the pool of membrane proteins deposited in PDB. Despite these advances, crystallization of membrane proteins still remains the main bottleneck in obtaining high quality structures. Recently, the use of antibody and non-antibody scaffold binding partners as crystallization “chaperones” has emerged as a powerful method to obtaining well-diffracting crystals of membrane proteins. In this chapter, a protocol is provided to generate synthetic antibody fragments for use as crystallization chaperones for membrane proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 159.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Wallin E, Heijne GV (1998) Genome‐wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci 7:1029–1038. doi:10.1002/pro.5560070420

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Coste B, Mathur J, Schmidt M et al (2010) Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330:55–60. doi:10.1126/science.1193270

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Hubbard R, Kropf A (1958) The action of light on rhodopsin. Proc Natl Acad Sci U S A. doi:10.1126/science.1193270

    PubMed  PubMed Central  Google Scholar 

  4. Catterall AW (1995) Structure and function of voltage-gated ion channels. Annu Rev Biochem 64:493–531. doi:10.1146/annurev.bi.64.070195.002425

    Article  CAS  PubMed  Google Scholar 

  5. Privé GG (2007) Detergents for the stabilization and crystallization of membrane proteins. Methods. doi:10.1146/annurev.bi.55.070186.004513

    PubMed  Google Scholar 

  6. Seddon AM, Curnow P, Booth PJ (2004) Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta 1666:105–117. doi:10.1016/j.bbamem.2004.04.011

    Article  CAS  PubMed  Google Scholar 

  7. Michel H (1983) Crystallization of membrane proteins. Trends Biochem Sci 8:56–59. doi:10.1016/0968-0004(83)90390-0

    Article  CAS  Google Scholar 

  8. Caffrey M, Li D, Dukkipati A (2012) Membrane protein structure determination using crystallography and lipidic mesophases: recent advances and successes. Biochemistry 51:6266–6288. doi:10.1021/bi300010w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Faham S, Bowie JU (2002) Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. J Mol Biol 316:1–6. doi:10.1006/jmbi.2001.5295

    Article  CAS  PubMed  Google Scholar 

  10. Iwata S, Ostermeier C, Ludwig B, Michel H (1995) Structure at 2.8 Å resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature 376:660–669

    Article  CAS  PubMed  Google Scholar 

  11. Zhou Y, Morais-Cabral JH, Kaufman A, MacKinnon R (2001) Chemistry of ion coordination and hydration revealed by a K+ channel–Fab complex at 2.0 Å resolution. Nature 414:43–48. doi:10.1038/35102009

    Article  CAS  PubMed  Google Scholar 

  12. Hino T, Arakawa T, Iwanari H et al (2012) G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody. Nature 482:237–240. doi:10.1038/nature10750

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Fang Y, Jayaram H, Shane T et al (2009) Structure of a prokaryotic virtual proton pump at 3.2 Å resolution. Nature 460:1040–1043. doi:10.1038/nature08201

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317. doi:10.1126/science.4001944

    Article  CAS  PubMed  Google Scholar 

  15. Boder ET, Wittruo KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557. doi:10.1038/nbt0697-553

    Article  CAS  PubMed  Google Scholar 

  16. Hanes J, Plückthun A (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci U S A. doi:10.1038/nbt.1791

    PubMed  PubMed Central  Google Scholar 

  17. Jostock T, Dübel S (2005) Screening of molecular repertoires by microbial surface display. Comb Chem High Throughput Screen 8:127–133. doi:10.2174/1386207053258479

    Article  CAS  PubMed  Google Scholar 

  18. de Haard HJ, van Neer N, Reurs A et al (1999) A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 274:18218–18230

    Article  PubMed  Google Scholar 

  19. Vaughan TJ, Williams AJ, Pritchard K et al (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 14:309–314. doi:10.1038/nbt0396-309

    Article  CAS  PubMed  Google Scholar 

  20. Arbabi Ghahroudi M, Desmyter A, Wyns L et al (1998) Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett 414:521–526. doi:10.1016/S0014-5793(97)01062-4

    Article  Google Scholar 

  21. Binz HK, Amstutz P, Kohl A et al (2004) High-affinity binders selected from designed ankyrin repeat protein libraries. Nat Biotechnol 22:575–582. doi:10.1038/nbt962

    Article  CAS  PubMed  Google Scholar 

  22. Koide A, Bailey CW, Huang X, Koide S (1998) The fibronectin type III domain as a scaffold for novel binding proteins. J Mol Biol 284:1141–1151. doi:10.1006/jmbi.1998.2238

    Article  CAS  PubMed  Google Scholar 

  23. Schönfeld D, Matschiner G, Chatwell L et al (2009) An engineered lipocalin specific for CTLA-4 reveals a combining site with structural and conformational features similar to antibodies. Proc Natl Acad Sci U S A 106:8198–8203. doi:10.1073/pnas.0813399106

    Article  PubMed  PubMed Central  Google Scholar 

  24. Cyranka-Czaja A, Otlewski J (2012) A novel, stable, helical scaffold as an alternative binder—construction of phage display libraries. Acta Biochim Pol 59(3):383–390

    CAS  PubMed  Google Scholar 

  25. Krishnamurthy H, Gouaux E (2012) X-ray structures of LeuT in substrate-free outward-open and apo inward-open states. Nature 481:469–474. doi:10.1038/nature10737

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Hino T, Iwata S, Murata T (2013) Generation of functional antibodies for mammalian membrane protein crystallography. Curr Opin Struct Biol 23:563–568. doi:10.1016/j.sbi.2013.04.007

    Article  CAS  PubMed  Google Scholar 

  27. Fellouse FA, Wiesmann C, Sidhu SS (2004) Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition. Proc Natl Acad Sci U S A 101:12467–12472. doi:10.1073/pnas.0401786101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ponsel D, Neugebauer J, Ladetzki-Baehs K, Tissot K (2011) High affinity, developability and functional size: the holy grail of combinatorial antibody library generation. Molecules 16:3675–3700. doi:10.3390/molecules16053675

    Article  CAS  PubMed  Google Scholar 

  29. Lee CV, Liang W-C, Dennis MS et al (2004) High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J Mol Biol 340:1073–1093. doi:10.1016/j.jmb.2004.05.051

    Article  CAS  PubMed  Google Scholar 

  30. Uysal S, Vásquez V, Tereshko V et al (2009) Crystal structure of full-length KcsA in its closed conformation. Proc Natl Acad Sci U S A 106:6644–6649. doi:10.1073/pnas.0810663106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ye J-D, Tereshko V, Frederiksen JK et al (2008) Synthetic antibodies for specific recognition and crystallization of structured RNA. Proc Natl Acad Sci U S A 105:82–87. doi:10.1073/pnas.0709082105

    Article  CAS  PubMed  Google Scholar 

  32. Razai A, Garcia-Rodriguez C, Lou J et al (2005) Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A. J Mol Biol 351:158–169. doi:10.1016/j.jmb.2005.06.003

    Article  CAS  PubMed  Google Scholar 

  33. Uysal S, Cuello LG, Cortes DM et al (2011) Mechanism of activation gating in the full-length KcsA K+ channel. Proc Natl Acad Sci U S A 108:11896–11899. doi:10.1073/pnas.1105112108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Li Q, Wanderling S, Paduch M et al (2014) Structural mechanism of voltage-dependent gating in an isolated voltage-sensing domain. Nat Struct Mol Biol 21:244–252. doi:10.1038/nsmb.2768

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Doyle DA, Cabral JM, Pfuetzner RA et al (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280:69–77. doi:10.1126/science.280.5360.69

    Article  CAS  PubMed  Google Scholar 

  36. Fellouse FA, Esaki K, Birtalan S et al (2007) High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J Mol Biol 373:924–940. doi:10.1016/j.jmb.2007.08.005

    Article  CAS  PubMed  Google Scholar 

  37. Fellouse FA, Barthelemy PA, Kelley RF, Sidhu SS (2006) Tyrosine plays a dominant functional role in the paratope of a synthetic antibody derived from a four amino acid code. J Mol Biol 357:100–114. doi:10.1016/j.jmb.2005.11.092

    Article  CAS  PubMed  Google Scholar 

  38. Birtalan S, Zhang Y, Fellouse FA et al (2008) The intrinsic contributions of tyrosine, serine, glycine and arginine to the affinity and specificity of antibodies. J Mol Biol 377:1518–1528. doi:10.1016/j.jmb.2008.01.093

    Article  CAS  PubMed  Google Scholar 

  39. Adams JJ, Nelson B, Sidhu SS (2014) Recombinant genetic libraries and human monoclonal antibodies. Methods Mol Biol 1060:149–170. doi:10.1007/978-1-62703-586-6_9

    Article  PubMed  Google Scholar 

  40. Paduch M, Koide A, Uysal S et al (2013) Generating conformation-specific synthetic antibodies to trap proteins in selected functional states. Methods 60:3–14. doi:10.1016/j.ymeth.2012.12.010

    Article  CAS  PubMed  Google Scholar 

  41. Zhong N, Loppnau P, Seitova A et al (2015) Optimizing production of antigens and Fabs in the context of generating recombinant antibodies to human proteins. PLoS One 10:e0139695. doi:10.1371/journal.pone.0139695

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Basic Medical Sciences, School of Medicine, Bezmialem Vakif University, Istanbul, Turkey

    Serdar Uysal

  2. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 60637, USA

    Anthony Kossiakoff

Authors
  1. Serdar Uysal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anthony Kossiakoff
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Serdar Uysal .

Editor information

Editors and Affiliations

  1. Indian Institute of Technology, Kanpur, India

    Arun K. Shukla

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Uysal, S., Kossiakoff, A. (2017). Generation of Synthetic Antibody Fragments to Detergent Solubilized Membrane Proteins. In: Shukla, A. (eds) Chemical and Synthetic Approaches in Membrane Biology. Springer Protocols Handbooks. Humana Press, New York, NY. https://doi.org/10.1007/8623_2016_11

Download citation

  • DOI: https://doi.org/10.1007/8623_2016_11

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6835-0

  • Online ISBN: 978-1-4939-6836-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation