Journal of Biomolecular NMR

, Volume 53, Issue 1, pp 43–51 | Cite as

High-yield Escherichia coli-based cell-free expression of human proteins



Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease activity. In the implementation of the expression system we used freshly in-house prepared cell extract. Batch-mode cell-free expression with this setup was up to twofold more economical than continuous-exchange expression, with yields of 0.2–0.9 mg of purified protein per mL of reaction mixture. Native folding of the proteins thus obtained is documented with 2D [15N,1H]-HSQC NMR.


Batch-mode cell-free protein expression Escherichia coli S30 cell extract Stable-isotope labeling Structural biology of human proteins 





Batch mode cell-free


Base pairs


Continuous-exchange cell-free






Dihydrofolate reductase




Ethylenediaminetetraacetic acid


Adipocyte fatty acid-binding protein 4


Human peptidyl-prolyl cis–trans isomerase FKBP1A


B1 domain of protein G from Streptococcus sp


γ-Interferon-inducible lysosomal thiol reductase


Heteronuclear single quantum coherence




Luria Bertani


Mitochondrial methylmalonyl-CoA epimerase


Methenyl-THF synthetase




Phosphate-buffered saline


Purkinje cell protein 2 homolog


Peptidyl-prolyl cis–trans isomerase B


Phosphate/yeast extract/glucose


Sodium dodecyl-sulfate polyacrylamide gel electrophoresis


Stromal cell-derived growth factor


RNA polymerase from bacteriophage T7


Tris-buffered saline



We thank Cristina Stocker for help with cell extract preparations, Dr. Arthur Horwich for providing the pET21-DHFR plasmid, Dr. Eilika Weber-Ban for providing the pET19-TEV plasmid, and the Swiss National Science Foundation and the ETH Zürich for financial support through the National Center of Competence in Research (NCCR) Structural Biology.

Supplementary material

10858_2012_9619_MOESM1_ESM.docx (28 kb)
Supplementary material 1 (DOCX 29 kb)
10858_2012_9619_MOESM2_ESM.tif (5.4 mb)
Supplementary material 2 (TIFF 5567 kb)


  1. Anderson CW, Straus JW, Dudock BS (1983) Preparation of a cell-free protein-synthesizing system from wheat germ. Methods Enzymol 101:635–644CrossRefGoogle Scholar
  2. Butt TR, Jonnalagadda S, Monia BP, Sternberg EJ, Marsh JA, Stadel JM, Ecker DJ, Crooke ST (1989) Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli. Proc Natl Acad Sci USA 86:2540–2544ADSCrossRefGoogle Scholar
  3. Chekulayeva MN, Kurnasov OV, Shirokov VA, Spirin AS (2001) Continuous-exchange cell-free protein-synthesizing system: synthesis of HIV-1 antigen Nef. Biochem Biophys Res Commun 280:914–917CrossRefGoogle Scholar
  4. Chen HZ, Zubay G (1983) Prokaryotic coupled transcription-translation. Methods Enzymol 101:674–690Google Scholar
  5. Chen X, Tomchick DR, Kovrigin E, Arac D, Machius M, Sudhof TC, Rizo J (2002) Three-dimensional structure of the complexin/SNARE complex. Neuron 33:397–409CrossRefGoogle Scholar
  6. Dyson HJ, Wright PE (2005) Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol 6:197–208CrossRefGoogle Scholar
  7. Endo Y, Otsuzuki S, Ito K, Miura K (1992) Production of an enzymatic active protein using a continuous flow cell-free translation system. J Biotechnol 25:221–230CrossRefGoogle Scholar
  8. Etezady-Esfarjani T, Hiller S, Villalba C, Wüthrich K (2007) Cell-free protein synthesis of perdeuterated proteins for NMR studies. J Biomol NMR 39:229–238CrossRefGoogle Scholar
  9. Ezure T, Suzuki T, Higashide S, Shintani E, Endo K, Kobayashi S, Shikata M, Ito M, Tanimizu K, Nishimura O (2006) Cell-free protein synthesis system prepared from insect cells by freeze-thawing. Biotechnol Prog 22:1570–1577CrossRefGoogle Scholar
  10. Goerke AR, Swartz JR (2008) Development of cell-free protein synthesis platforms for disulfide bonded proteins. Biotechnol Bioeng 99:351–367CrossRefGoogle Scholar
  11. Güntert P, Dötsch V, Wider G, Wüthrich K (1992) Processing of multi-dimensional NMR data with the new software PROSA. J Biomol NMR 2:619–629CrossRefGoogle Scholar
  12. Henshaw EC, Panniers R (1983) Translational systems prepared from the Ehrlich ascites tumor cell. Methods Enzymol 101:616–629CrossRefGoogle Scholar
  13. Jermutus L, Ryabova LA, Pluckthun A (1998) Recent advances in producing and selecting functional proteins by using cell-free translation. Curr Opin Biotechnol 9:534–548CrossRefGoogle Scholar
  14. Kawasaki T, Gouda MD, Sawasaki T, Takai K, Endo Y (2003) Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ. Eur J Biochem 270:4780–4786CrossRefGoogle Scholar
  15. Keller R (2004) The computer aided resonance assignment tutorial. Goldau, CantinaGoogle Scholar
  16. Kigawa T, Yabuki T, Matsuda N, Matsuda T, Nakajima R, Tanaka A, Yokoyama S (2004) Preparation of Escherichia coli cell extract for highly productive cell-free protein expression. J Struct Funct Genom 5:63–68CrossRefGoogle Scholar
  17. Kigawa T, Matsuda T, Yabuki T, Yokoyama S (2008) Baterial cell-free system for highly efficient protein synthesis. Wiley-VCH, WeinheimGoogle Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685ADSCrossRefGoogle Scholar
  19. Laursen BS, Sorensen HP, Mortensen KK, Sperling-Petersen HU (2005) Initiation of protein synthesis in bacteria. Microbiol Mol Biol Rev 69:101–123CrossRefGoogle Scholar
  20. Linding R, Russell RB, Neduva V, Gibson TJ (2003) GlobPlot: exploring protein sequences for globularity and disorder. Nucleic Acids Res 31:3701–3708CrossRefGoogle Scholar
  21. Marr E, Tardie M, Carty M, Brown Phillips T, Wang IK, Soeller W, Qiu X, Karam G (2006) Expression, purification, crystallization and structure of human adipocyte lipid-binding protein (aP2). Acta Crystallogr Sect F Struct Biol Cryst Commun 62:1058–1060CrossRefGoogle Scholar
  22. Michel-Reydellet N, Calhoun K, Swartz J (2004) Amino acid stabilization for cell-free protein synthesis by modification of the Escherichia coli genome. Metab Eng 6:197–203CrossRefGoogle Scholar
  23. Mikol V, Kallen J, Walkinshaw MD (1994) X-ray structure of a cyclophilin B/cyclosporin complex: comparison with cyclophilin A and delineation of its calcineurin-binding domain. Proc Natl Acad Sci USA 91:5183–5186ADSCrossRefGoogle Scholar
  24. Nirenberg MW, Matthaei JH (1961) The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci USA 47:1588–1602Google Scholar
  25. Ozawa K, Headlam MJ, Schaeffer PM, Henderson BR, Dixon NE, Otting G (2004) Optimization of an Escherichia coli system for cell-free synthesis of selectively N-labelled proteins for rapid analysis by NMR spectroscopy. Eur J Biochem 271:4084–4093CrossRefGoogle Scholar
  26. Pratt JM (1984) Coupled transcription-translation in prokaryotic cell-free systems. Oxford University Press, OxfordGoogle Scholar
  27. Schwarz D, Junge F, Durst F, Frölich N, Schneider B, Reckel S, Sobhanifar S, Dötsch V, Bernhard F (2007) Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems. Nat Protoc 2:2945–2957CrossRefGoogle Scholar
  28. Sitaraman K, Chatterjee DK (2009) High-throughput protein expression using cell-free system. Methods Mol Biol High Throughput Protein Express Purif 498:229–244CrossRefGoogle Scholar
  29. Spirin AS (2004) High-throughput cell-free systems for synthesis of functionally active proteins. Trends Biotechnol 22:538–545CrossRefGoogle Scholar
  30. Staunton D, Schlinkert R, Zanetti G, Colebrook SA, Campbell ID (2006) Cell-free expression and selective isotope labelling in protein NMR. Magn Reson Chem 44:S2–S9Google Scholar
  31. Stockman BJ, Nirmala NR, Wagner G, Delcamp TJ, Deyarman MT, Freisheim JH (1992) Sequence-specific 1H and 15 N resonance assignments for human dihydrofolate reductase in solution. Biochemistry 31:218–229CrossRefGoogle Scholar
  32. Torizawa T, Shimizu M, Taoka M, Miyano H, Kainosho M (2004) Efficient production of isotopically labeled proteins by cell-free synthesis: a practical protocol. J Biomol NMR 30:311–325CrossRefGoogle Scholar
  33. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354ADSCrossRefGoogle Scholar
  34. Vinarov DA, Loushin Newman CL, Markley JL (2006) Wheat germ cell-free platform for eukaryotic protein production. FEBS J 273:4160–4169CrossRefGoogle Scholar
  35. Xu Z, Chen H, Yin X, Xu N, Cen P (2005) High-level expression of soluble human beta-defensin-2 fused with green fluorescent protein in Escherichia coli cell-free system. Appl Biochem Biotechnol 127:53–62CrossRefGoogle Scholar
  36. Zhou P, Lugovskoy AA, Wagner G (2001) A solubility-enhancement tag (SET) for NMR studies of poorly behaving proteins. J Biomol NMR 20:11–14CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  1. 1.Institute of Molecular Biology and BiophysicsETH ZurichZurichSwitzerland

Personalised recommendations