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High-level expression of soluble subunit b of F1F0 ATP synthase in Escherichia coli cell-free system

Abstract

The overexpression of subunit b of F1F0 adenosine triphosphate (ATP) synthase from Escherichia coli is so toxic that it even prevents the transformation of plasmids encoding this protein into E. coli BL21 (DE3). In the present work, E. coli cell-free system was chosen as an alternative to express this highly toxic membrane protein. This protein was either produced as precipitates followed by detergent resolubilization or expressed as a soluble form with detergent addition. Among several types of tested detergents, Brij 58 could effectively solubilize approximately 85% of the target membrane protein within a wide range of concentration (48 to 178 times critical micelle concentration [CMC]) with little effect on the expression level. With the presence of Brij 58 at the final concentration of 96 times CMC in the E. coli cell-free system, 789 μg/mL of soluble subunit b was achieved after 4 h biosynthesis, which is the highest level for the expression of membrane proteins in a batch-mode cell-free expression system. The present work provides a rapid and efficient procedure of expressing one membrane protein with high cytotoxicity in the cell-free system and will be helpful to further exploration of reconstituting F1F0 ATP synthase into liposome or polymer vesicle to design a nanoelectromechanical system device.

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References

  1. Arechaga I, Miroux B, Karrasch S, Huijbregts R, Kruijff BD, Runswick MJ, Walker JE (2000) Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F1F0 ATP synthase. FEBS Lett 482:215–219

  2. Berrier C, Park KH, Abes S, Bibonne A, Betton JM, Ghazi A (2004) Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent. Biochemistry 43:12585–12591

  3. Boyer PD (1997) The ATP synthase—a splendid molecular machine. Annu Rev Biochem 66:717–749

  4. Chen HQ, Xu ZN, Xu NZ, Cen PL (2005) Efficient production of a soluble fusion protein containing human beta-defensin-2 in E. coli cell-free system. J Biotechnol 115:307–315

  5. Chen HQ, Xu ZN, Xu NZ, Cen PL (2006) High-level expression of human-β-defensin-2 gene with rare codons in E. coli cell-free system. Protein Pept Lett 13:155–161

  6. Chen HQ, Fan LM, Xu ZN, Yin XF, Cen PL (2007) Efficient production of soluble beta-defensin-3–4 fusion proteins in Escherichia coli cell-free system. Process Biochem 42:423–428

  7. DeGrip WJ (1982) Thermal stability of rhodopsin and opsin in some novel detergents. Methods Enzymol 81:256–265

  8. Elbaz Y, Steiner-Mordoch S, Danieli T, Schuldiner S (2004) In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state. Proc Natl Acad Sci U S A 101:1519–1524

  9. Fillinggame RH, Jones PC, Jiang W, Valiyaveetil FI, Dmitriev OY (1998) Subunit organization and structure in the F0 sector of Escherichia coli F1F0 ATP synthase. Biochim Biophys Acta 1365:135–142

  10. Giorno L, Drioli E (2000) Biocatalytic membrane reactors: applications and perspectives. Trends Biotechnol 18:339–349

  11. Goerke AR, Swartz JR (2008) Development of cell-free protein synthesis platforms for disulfide bonded proteins. Biotechnol Bioeng 99:351–367

  12. Ishihara G, Goto M, Saeki M, Ito K, Hori T, Kigawa T, Shirouzu M, Yokoyama S (2005) Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors. Protein Expr Purif 41:27–37

  13. Kalmbach R, Chizhov I, Schumacher MC, Friedrich T, Bamberg E, Engelhard M (2007) Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes. J Mol Biol 371:639–648

  14. Katzen F, Chang G, Kudlicki W (2005) The past, present and future of cell-free protein synthesis. Trends Biotechol 23:150–156

  15. Kiefer H, Krieger J, Olszewski JD, Heijne G, Prestwich GD, Breer H (1996) Expression of an olfactory receptor in Escherichia coli: purification, reconstitution, and ligand binding. Biochemistry 35(3):16077–16084

  16. Klammt C, Lohr F, Schafer B, Haase W, Dotsch V, Ruterjans GC, Bernhard F (2004) High level cell-free expression and specific labeling of integral membrane proteins. Eur J Biochem 271:568–580

  17. Klammt C, Schwarz D, Fendler K, Haase W, Dotsch V, Bernhard F (2005) Evaluation of detergents for the soluble expression of α-helical and β-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system. FEBS J 272:6024–6038

  18. Klammt C, Schwarz D, Lohr F, Schneider B, Dotsch V, Bernhard F (2006) Cell-free expression as an emerging technique for the large scale production of integral membrane protein. FEBS J 273:4141–4153

  19. Klammt C, Schwarz D, Eifler N, Engel A, Piehler J, Haase W, Hahn S, Dotsch V, Bernhard F (2007) Cell-free production of G protein-coupled receptors for functional and structural studies. J Struct Biol 158:482–493

  20. Lian JZ, Fang XM, Cai J, Chen QX, Zheng Q, Kai L, Xu ZN (2008) Efficient expression of membrane-bound water channel protein (Aquaporin Z) in Escherichia coli. Protein Pept Lett 15:687–691

  21. Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298

  22. Nagamori S, Vazquez-Ibar JL, Weinglass AB, Kaback HR (2003) In vitro synthesis of lactose permease to probe the mechanism of membrane insertion and folding. J Biol Chem 278:14820–14826

  23. Nakano H, Yamane T (1998) Cell-free protein synthesis systems. Biotechnol Adv 16:367–384

  24. Odahara T (2004) Stability and solubility of integral membrane proteins from photosynthetic bacteria solubilized in different detergents. Biochim Biophys Acta 1660:80–92

  25. Park KH, Berrier C, Lebaupain F, Pucci B, Popot JL, Ghazi A, Zito F (2007) Flurinated and hemiflurinated surfactants as alternatives to detergents for membrane protein cell-free synthesis. Biochem J 403:183–187

  26. Porter ACG, Kumamoto C, Aldape K, Simoni RD (1985) Role of the b subunit of the Escherichia coli proton-translocating ATPase: a mutagenic analysis. J Biol Chem 260:8182–8187

  27. Rungpragayphan S, Nakano H, Yamane T (2003) PCR-linked in vitro expression: a novel system for high-throughput construction and screening of protein libraries. FEBS Lett 540:147–150

  28. Sasaki S, Karube I (1999) The development of microfabricated biocatalytic fuel cells. Trends Biotechnol 17:50–52

  29. Schwarz D, Klammt C, Koglin A, Lohr F, Schneider B, Dotsch V, Bernhard F (2007) Preparative scale cell-free expression systems: new tools for the large scale preparation of integral membrane proteins for functional and structural studies. Methods 41:355–369

  30. Senior AE (1990) The proton-translocation ATPase of Escherichia coli. Annu Rev Biophys Biophys Chem 19:7–41

  31. Shim JW, Yang MM, Gu LQ (2007) In vitro synthesis, tetramerization and single channel characterization of virus-encoded potassium channel Kcv. FEBS Lett 581:1027–1034

  32. Shimada Y, Wang ZY, Mochizuki Y, Kobayashi M, Nozawa T (2004) Functional expression and characterization of a bacterial light-harvesting membrane protein in Escherichia coli and cell-free synthesis system. Biosci Biotechnol Biochem 68(9):1942–1948

  33. Soong RK, Bachand GD, Neves HP, Olkhovets AG, Craighead HG, Montemagno CD (2000) Powering an inorganic nanodevice with a biomolecular motor. Science 290:1555–1558

  34. Tate CG (2001) Overexpression of mammalian integral membrane proteins for structural studies. FEBS Lett 504(3):94–98

  35. Wang DN, Safferling M, Lemieux MJ, Griffith H, Chen Y, Li XD (2003) Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies. Biochim Biophys Acta 1610:23–36

  36. Wuu JJ, Swartz JR (2008) High yield cell-free production of integral membrane proteins without refolding or detergents. Biochim Biophys Acta 1778:1237–1250

  37. Xu ZN, Chen HQ, Yin XF, Xu NZ, Cen PL (2005) High-level expression of soluble human β-defensin-2 fused with green fluorescent protein in Escherichia coli cell-free system. Appl Biochem Biotechnol 127:53–62

  38. Yin G, Swartz JR (2004) Enhancing multiple disulfide bonded protein folding in a cell-free system. Biotechnol Bioeng 86:188–195

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Acknowledgements

This work was financially supported by The National Natural Science Foundation of China (grant nos. 20736008 and 20676115) and The Ministry of Science and Technology (grant nos. 2007AA021702 and 2009CB918600), The People's Republic of China.

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Correspondence to Zhinan Xu.

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Lian, J., Ma, Y., Cai, J. et al. High-level expression of soluble subunit b of F1F0 ATP synthase in Escherichia coli cell-free system. Appl Microbiol Biotechnol 85, 303–311 (2009). https://doi.org/10.1007/s00253-009-2055-z

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Keywords

  • Subunit b of ATP synthase
  • ATP synthase
  • Cell-free expression system
  • Membrane protein
  • Escherichia coli