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Liposomes Mediated Synthesis of Membrane Proteins

  • Yutetsu KurumaEmail author
Chapter

Abstract

Synthetic biology is an emerging field that aims at constructing artificial biological systems by combining engineering and molecular biology approaches. One of the most ambitious research line concerns the socalled semi-synthetic minimal cells, which are liposome-based system capable of synthesizing the lipids within the liposome surface. This goal can be reached by reconstituting membrane proteins within liposomes and allow them to synthesize lipids. This approach, that can be defined as biochemical, was already reported by Schmidli et al. (in 1991). In more advanced models, however, a full reconstruction of the biochemical pathway requires (1) the synthesis of functional membrane enzymes inside liposomes, and (2) the local synthesis of lipids as catalyzed by the in situ synthesized enzymes. Here we show the synthesis and the activity – inside liposomes – of two membrane proteins involved in phospholipids biosynthesis pathway. The proteins, sn-glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT), have been synthesized by using a totally reconstructed cell-free system (PURE system) encapsulated in liposomes. The activities of internally synthesized GPAT and LPAAT were confirmed by detecting the produced lysophosphatidic acid and phosphatidic acid, respectively. Through this procedure, we have implemented the first phase of a design aimed at synthesizing phospholipid membrane from liposome within – which corresponds to the autopoietic growth mechanism.

Keywords

Liposomes Membrane protein Cell-free translation system Lipid synthesis Minimal cells 

References

  1. Cronan JE (2003) Bacterial membrane lipids: where do we stand? Annu Rev Microbiol 57:203–224CrossRefPubMedGoogle Scholar
  2. Gil R, Silva FJ, Peretó J, Moya A (2004) Determination of the core of a minimal bacterial gene set. Microbiol Mol Biol Rev 68:518–537CrossRefPubMedGoogle Scholar
  3. Glass JI, Assad-Garcia N, Alperovich N, Yooseph S, Lewis MR, Maruf M, Hutchison CA 3rd, Smith HO, Venter JC (2006) Essential genes of a minimal bacterium. Proc Natl Acad Sci USA 103:425–430CrossRefPubMedGoogle Scholar
  4. Gold VA, Duong F, Collinson I (2007) Structure and function of the bacterial Sec translocon. Mol Membr Biol 24:387–394CrossRefPubMedGoogle Scholar
  5. Hutchison CA, Peterson SN, Gill SR, Cline RT, White O, Fraser CM, Smith HO, Venter JC (1999) Global transposon mutagenesis and a minimal Mycoplasma genome. Science 286:2165–2169CrossRefPubMedGoogle Scholar
  6. Ishikawa K, Sato K, Shima Y, Urabe I, Yomo T (2004) Expression of a cascading genetic network within liposomes. FEBS Lett 576:387–390CrossRefPubMedGoogle Scholar
  7. Jayasinghe L, Miles G, Bayley H (2006) Role of the amino latch of staphylococcal alpha-hemolysin in pore formation: a co-operative interaction between the N terminus and position 217. J Biol Chem 281:2195–2204CrossRefPubMedGoogle Scholar
  8. Kawano R, Schibel AE, Cauley C, White HS (2009) Controlling the translocation of single-stranded DNA through alpha-hemolysin ion channels using viscosity. Langmuir 25:1233–1237CrossRefPubMedGoogle Scholar
  9. Kawashima Y, Miyazaki E, Müller M, Tokuda H, Nishiyama K (2008) Diacylglycerol specifically blocks spontaneous integration of membrane proteins and allows detection of a factor-assisted integration. J Biol Chem 283:24489–24496CrossRefPubMedGoogle Scholar
  10. Kita H, Matsuura T, Sunami T, Hosoda K, Ichihashi N, Tsukada K, Urabe I, Yomo T (2008) Replication of genetic information with self-encoded replicase in liposomes. Chembiochem 9:2403–2410CrossRefPubMedGoogle Scholar
  11. Kuruma Y, Stano P, Ueda T, Luisi PL (2009) A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells. Biochim Biophys Acta 1788:567–574CrossRefPubMedGoogle Scholar
  12. Kuruma Y, Nishiyama K, Shimizu Y, Müller M, Ueda T (2005) Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins. Biotechnol Prog 21:1243–1251CrossRefPubMedGoogle Scholar
  13. Luirink J, von Heijne G, Houben E, de Gier JW (2005) Biogenesis of inner membrane proteins in Escherichia coli. Annu Rev Microbiol 59:329–355CrossRefPubMedGoogle Scholar
  14. Luisi PL (2006) The emergence of life. From chemical origins to synthetic biology. Cambridge University Press, CambridgeGoogle Scholar
  15. Luisi PL (2007) Chemical aspects of synthetic biology. Chem Biodivers 4:603–621CrossRefPubMedGoogle Scholar
  16. Luisi PL, Ferri F, Stano P (2006) Approaches to semi-synthetic minimal cells. Naturwissenschaften 93:1–13CrossRefPubMedGoogle Scholar
  17. Mushegian AR, Koonin EV (1996) A minimal gene set for cellular life derived by comparison of complete bacterial genomes. Proc Natl Acad Sci USA 93:10268–10273CrossRefPubMedGoogle Scholar
  18. Moya A, Gil R, Latorre A, Peretó J, Pilar Garcillán-Barcia M, de la Cruz F (2009) Toward minimal bacterial cells: evolution vs. design. FEMS Microbiol Rev 33:225–235CrossRefPubMedGoogle Scholar
  19. Nomura SM, Tsumoto K, Hamada T, Akiyoshi K, Nakatani Y, Yoshikawa K (2003) Gene expression within cell-sized lipid vesicles. Chembiochem 4:1172–1175Google Scholar
  20. Nomura SM, Kondoh S, Asayama W, Asada A, Nishikawa S, Akiyoshi K (2008) Direct preparation of giant proteo-liposomes by in vitro membrane protein synthesis. J Biotechnol 133:190–195CrossRefPubMedGoogle Scholar
  21. Noireaux V, Libchaber A (2004) A vesicle bioreactor as a step toward an artificial cell assembly. Proc Natl Acad Sci USA 101:17669–17674CrossRefPubMedGoogle Scholar
  22. Oberholzer T, Luisi PL (2002) The use of liposomes for constructing cell models. J Biol Phys 28:733–744CrossRefGoogle Scholar
  23. Schmidli PK, Schurtenberger P, Luisi PL (1991) Liposome-mediated enzymatic synthesis of phosphatidylcholine as an approach to self-replicating liposomes. J Am Chem Soc 113:8127–8130Google Scholar
  24. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T (2001) Cell-free translation reconstituted with purified components. Nat Biotechnol 19:751–755CrossRefPubMedGoogle Scholar
  25. Sunami T, Sato K, Matsuura T, Tsukada K, Urabe I, Yomo T (2006) Femtoliter compartment in liposomes for in vitro selection of proteins. Anal Biochem 357:128–136CrossRefPubMedGoogle Scholar
  26. Tsukazaki T, Mori H, Fukai S, Ishitani R, Mori T, Dohmae N, Perederina A, Sugita Y, Vassylyev DG, Ito K, Nureki O (2008) Conformational transition of Sec machinery inferred from bacterial SecYE structures. Nature 455:988–991CrossRefPubMedGoogle Scholar
  27. Yu W, Sato K, Wakabayashi M, Nakaishi T, Ko-Mitamura EP, Shima Y, Urabe I, Yomo T (2001) Synthesis of functional protein in liposome. J Biosci Bioeng 92:590–593CrossRefPubMedGoogle Scholar

Copyright information

© Springer Netherlands 2011

Authors and Affiliations

  1. 1.Department of Medical Genome Sciences, Graduate School of Frontier SciencesThe University of TokyoKashiwaJapan

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