Production of Immunizing Antigen Proteoliposome Using Cell-Free Protein Synthesis System

  • Wei Zhou
  • Hiroyuki TakedaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1868)


Antibodies specifically recognizing integral membrane protein are essential tool for functional analysis, diagnosis, and therapeutics targeting membrane proteins. However, development of antibodies against membrane protein remains a big challenge, because mass production of membrane protein is difficult. Recently, we developed a highly efficient cell-free production method of proteoliposome antigen using cell-free protein synthesis method with liposome and dialysis cup. Here we introduce practical and efficient integrated procedures to produce large amount of proteoliposome antigen for anti-membrane protein antibody development.

Key words

Proteoliposome Membrane protein Cell-free protein synthesis Immunizing antigen Adjuvant 



The authors thank Mr. Tomio Ogasawara for his assistance in the technological development. We also thank Professor Tatsuya Sawasaki for his mentoring. This work was mainly supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED, Japan. This work was also partially supported by JSPS KAKENHI Grant Numbers 24710251 and 26750375 and 16k01915.


  1. 1.
    Wilkinson TCI (2016) Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels. Biochem Soc Trans 44:831–837. Scholar
  2. 2.
    Hino T, Iwata S, Murata T (2013) Generation of functional antibodies for mammalian membrane protein crystallography. Curr Opin Struct Biol 23:563–568. Scholar
  3. 3.
    Webb DR, Handel TM, Kretz-Rommel A, Stevens RC (2013) Opportunities for functional selectivity in GPCR antibodies. Biochem Pharmacol 85:147–152. Scholar
  4. 4.
    Ecker DM, Jones SD, Levine HL (2015) The therapeutic monoclonal antibody market. MAbs 7:9–14. Scholar
  5. 5.
    Hutchings CJ, Koglin M, Marshall FH (2010) Therapeutic antibodies directed at G protein-coupled receptors. MAbs 2:594–606. Scholar
  6. 6.
    Hino T, Arakawa T, Iwanari H et al (2012) G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody. Nature 482:1–5. Scholar
  7. 7.
    Pone EJ, Zhang J, Mai T et al (2012) BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-κB pathway. Nat Commun 3:767. Scholar
  8. 8.
    Bill RM, Henderson PJF, Iwata S et al (2011) Overcoming barriers to membrane protein structure determination. Nat Biotechnol 29:335–340. Scholar
  9. 9.
    Seddon AM, Curnow P, Booth PJ (2004) Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta 1666:105–117. Scholar
  10. 10.
    Dalibor Milić DBV (2015) Large-scale production and protein engineering of G protein-coupled receptors for structural studies. Front Pharmacol 6:394. Scholar
  11. 11.
    Nozawa A, Ogasawara T, Matsunaga S et al (2011) Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system. BMC Biotechnol 11:35. Scholar
  12. 12.
    Suzuki Y, Ogasawara T, Tanaka Y et al (2018) Functional G-protein-coupled receptor (GPCR) synthesis: the pharmacological analysis of human histamine H1 receptor (HRH1) synthesized by a wheat germ cell-free protein synthesis system combined with asolectin glycerosomes. Front Pharmacol 9:38. Scholar
  13. 13.
    Sackin H, Nanazashvili M, Makino S-I (2015) Direct injection of cell-free Kir1.1 protein into Xenopus oocytes replicates single-channel currents derived from Kir1.1 mRNA. Channels 9:196–199. Scholar
  14. 14.
    Renauld S, Cortes S, Bersch B et al (2017) Functional reconstitution of cell-free synthesized purified Kv channels. Biochim Biophys Acta 1859:2373–2380. Scholar
  15. 15.
    Liu S, Hasegawa H, Takemasa E et al (2017) Efficiency and safety of CRAC inhibitors in human rheumatoid arthritis xenograft models. J Immunol 199:1584–1595. Scholar
  16. 16.
    Hashimoto Y, Zhou W, Hamauchi K, et al (2018) Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5. Sci Rep 8:8383. Scholar
  17. 17.
    Takeda H, Ogasawara T, Ozawa T et al (2015) Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay. Sci Rep 5:11,333. Scholar
  18. 18.
    Gibson DG (2011) Enzymatic assembly of overlapping DNA fragments. Methods Enzymol 498:349–361. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Proteo-Science CenterEhime UniversityMatsuyamaJapan

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