SpringerLink
Log in

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

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

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.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

2-ME:

β-Mercaptoethanol

BMCF:

Batch mode cell-free

bp:

Base pairs

CECF:

Continuous-exchange cell-free

kDa:

Kilo-Dalton

DEPC:

Diethylpyrocarbonate

DHFR:

Dihydrofolate reductase

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

FABP4:

Adipocyte fatty acid-binding protein 4

FKBP:

Human peptidyl-prolyl cis–trans isomerase FKBP1A

GB1:

B1 domain of protein G from Streptococcus sp

GILT:

γ-Interferon-inducible lysosomal thiol reductase

HSQC:

Heteronuclear single quantum coherence

IPTG:

Isopropyl-β-D-thiogalactopyranoside

LB:

Luria Bertani

MMCE:

Mitochondrial methylmalonyl-CoA epimerase

MTHFS:

Methenyl-THF synthetase

OAc:

Acetate

PBS:

Phosphate-buffered saline

PCP2H:

Purkinje cell protein 2 homolog

ppiB:

Peptidyl-prolyl cis–trans isomerase B

PYG:

Phosphate/yeast extract/glucose

SDS-PAGE:

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis

SF20:

Stromal cell-derived growth factor

T7 RNAP:

RNA polymerase from bacteriophage T7

TBS:

Tris-buffered saline

References

  • Anderson CW, Straus JW, Dudock BS (1983) Preparation of a cell-free protein-synthesizing system from wheat germ. Methods Enzymol 101:635–644

    Article  Google Scholar 

  • 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 86:2540–2544

    Article  ADS  Google Scholar 

  • 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 280:914–917

    Article  Google Scholar 

  • Chen HZ, Zubay G (1983) Prokaryotic coupled transcription-translation. Methods Enzymol 101:674–690

    Google Scholar 

  • Chen X, Tomchick DR, Kovrigin E, Arac D, Machius M, Sudhof TC, Rizo J (2002) Three-dimensional structure of the complexin/SNARE complex. Neuron 33:397–409

    Article  Google Scholar 

  • Dyson HJ, Wright PE (2005) Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol 6:197–208

    Article  Google Scholar 

  • 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 25:221–230

    Article  Google Scholar 

  • Etezady-Esfarjani T, Hiller S, Villalba C, Wüthrich K (2007) Cell-free protein synthesis of perdeuterated proteins for NMR studies. J Biomol NMR 39:229–238

    Article  Google Scholar 

  • 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 22:1570–1577

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Güntert P, Dötsch V, Wider G, Wüthrich K (1992) Processing of multi-dimensional NMR data with the new software PROSA. J Biomol NMR 2:619–629

    Article  Google Scholar 

  • Henshaw EC, Panniers R (1983) Translational systems prepared from the Ehrlich ascites tumor cell. Methods Enzymol 101:616–629

    Article  Google Scholar 

  • Jermutus L, Ryabova LA, Pluckthun A (1998) Recent advances in producing and selecting functional proteins by using cell-free translation. Curr Opin Biotechnol 9:534–548

    Article  Google Scholar 

  • 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 270:4780–4786

    Article  Google Scholar 

  • Keller R (2004) The computer aided resonance assignment tutorial. Goldau, Cantina

    Google Scholar 

  • 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 5:63–68

    Article  Google Scholar 

  • Kigawa T, Matsuda T, Yabuki T, Yokoyama S (2008) Baterial cell-free system for highly efficient protein synthesis. Wiley-VCH, Weinheim

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  ADS  Google Scholar 

  • Laursen BS, Sorensen HP, Mortensen KK, Sperling-Petersen HU (2005) Initiation of protein synthesis in bacteria. Microbiol Mol Biol Rev 69:101–123

    Article  Google Scholar 

  • Linding R, Russell RB, Neduva V, Gibson TJ (2003) GlobPlot: exploring protein sequences for globularity and disorder. Nucleic Acids Res 31:3701–3708

    Article  Google Scholar 

  • 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 62:1058–1060

    Article  Google Scholar 

  • 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 6:197–203

    Article  Google Scholar 

  • 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 91:5183–5186

    Article  ADS  Google Scholar 

  • 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 47:1588–1602

    Google Scholar 

  • 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 271:4084–4093

    Article  Google Scholar 

  • Pratt JM (1984) Coupled transcription-translation in prokaryotic cell-free systems. Oxford University Press, Oxford

    Google Scholar 

  • 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 2:2945–2957

    Article  Google Scholar 

  • Sitaraman K, Chatterjee DK (2009) High-throughput protein expression using cell-free system. Methods Mol Biol High Throughput Protein Express Purif 498:229–244

    Article  Google Scholar 

  • Spirin AS (2004) High-throughput cell-free systems for synthesis of functionally active proteins. Trends Biotechnol 22:538–545

    Article  Google Scholar 

  • 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–S9

    Google Scholar 

  • 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 31:218–229

    Article  Google Scholar 

  • 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 30:311–325

    Article  Google Scholar 

  • 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 76:4350–4354

    Article  ADS  Google Scholar 

  • Vinarov DA, Loushin Newman CL, Markley JL (2006) Wheat germ cell-free platform for eukaryotic protein production. FEBS J 273:4160–4169

    Article  Google Scholar 

  • 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 127:53–62

    Article  Google Scholar 

  • Zhou P, Lugovskoy AA, Wagner G (2001) A solubility-enhancement tag (SET) for NMR studies of poorly behaving proteins. J Biomol NMR 20:11–14

    Article  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. Institute of Molecular Biology and Biophysics, ETH Zurich, 8093, Zurich, Switzerland

    Erich Michel & Kurt Wüthrich

Authors
  1. Erich Michel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kurt Wüthrich
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kurt Wüthrich.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 29 kb)

Supplementary material 2 (TIFF 5567 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Michel, E., Wüthrich, K. High-yield Escherichia coli-based cell-free expression of human proteins. J Biomol NMR 53, 43–51 (2012). https://doi.org/10.1007/s10858-012-9619-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-012-9619-4

Keywords

Search

Navigation