Skip to main content
SpringerLink
Log in

Cell-free expression and stable isotope labelling strategies for membrane proteins

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

Abstract

Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems provides a very interesting alternative to cell-based systems, since it circumvents many problems such as toxicity or necessity for the transportation of the synthesized protein to the membrane, and constitutes the only system that allows for direct production of membrane proteins in membrane-mimetic environments which may be suitable for liquid state NMR measurements. The unique advantages of the cell-free expression system, including strong expression yields as well as the direct incorporation of almost any combination of amino acids with very little metabolic scrambling, has allowed for the development of a wide-array of isotope labelling techniques which facilitate structural investigations of proteins whose spectral congestion and broad line-widths may have earlier rendered them beyond the scope of NMR. Here we explore various labelling strategies in conjunction with cell-free developments, with a particular focus on α-helical transmembrane proteins which benefit most from such methods.

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

Access this article

Log in via an institution

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

Similar content being viewed by others

References

  • Arora A, Abildgaard F, Bushweller JH, Tamm LK (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat Struct Biol 8:334–338

    Article  Google Scholar 

  • Bain JD, Diala ES, Glabe CG, Wacker DA, Lyttle MH, Dix TA, Chamberlin AR (1991) Site-specific incorporation of nonnatural residues during in vitro protein biosynthesis with semisynthetic aminoacyl-tRNAs. Biochemistry 30:5411–5421

    Article  Google Scholar 

  • Bayrhuber M, Meins T, Habeck M, Becker S, Giller K, Villinger S, Vonrhein C, Griesinger C, Zweckstetter M, Zeth K (2008) Structure of the human voltage-dependent anion channel. Proc Natl Acad Sci USA 105:15370–15375

    Article  ADS  Google Scholar 

  • 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

    Article  Google Scholar 

  • Cappuccio JA, Blanchette CD, Sulchek TA, Arroyo ES, Kralj JM, Hinz AK, Kuhn EA, Chromy BA, Segelke BW, Rothschild KJ, Fletcher JE, Katzen F, Peterson TC, Kudlicki WA, Bench G, Hoeprich PD, Coleman MA (2008) Cell-free co-expression of functional membrane proteins and apolipoprotein, forming soluble nanolipoprotein particles. Mol Cell Proteomics 7:2246–2253

    Article  Google Scholar 

  • Chen YJ, Pornillos O, Lieu S, Ma C, Chen AP, Chang G (2007) X-ray structure of EmrE supports dual topology model. Proc Natl Acad Sci USA 104:18999–19004

    Article  ADS  Google Scholar 

  • Clore GM, Gronenborn AM (1998) NMR structure determination of proteins and protein complexes larger than 20 kDa. Curr Opin Chem Biol 2:564–570

    Article  Google Scholar 

  • 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 USA 101:1519–1524

    Article  ADS  Google Scholar 

  • Ellman JA, Volkman BF, Mendel D, Schulz PG, Wemmer DE (1992) Site-specific isotopic labeling of proteins for NMR studies. J Am Chem Soc 114:7959–7961

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Fernandez C, Hilty C, Wider G, Guntert P, Wuthrich K (2004) NMR structure of the integral membrane protein OmpX. J Mol Biol 336:1211–1221

    Article  Google Scholar 

  • Gardner KH, Kay LE (1998) The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins. Annu Rev Biophys Biomol Struct 27:357–406

    Article  Google Scholar 

  • Goerke AR, Swartz JR (2009) High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids. Biotechnol Bioeng 102:400–416

    Article  Google Scholar 

  • Goren MA, Fox BG (2008) Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex. Protein Expr Purif 62:171–178

    Article  Google Scholar 

  • Gourdon P, Alfredsson A, Pedersen A, Malmerberg E, Nyblom M, Widell M, Berntsson R, Pinhassi J, Braiman M, Hansson O, Bonander N, Karlsson G, Neutze R (2008) Optimized in vitro and in vivo expression of proteorhodopsin: a seven-transmembrane proton pump. Protein Expr Purif 58:103–113

    Article  Google Scholar 

  • Guignard L, Ozawa K, Pursglove SE, Otting G, Dixon NE (2002) NMR analysis of in vitro-synthesized proteins without purification: a high-throughput approach. FEBS Lett 524:159–162

    Article  Google Scholar 

  • Hiller S, Garces RG, Malia TJ, Orekhov VY, Colombini M, Wagner G (2008) Solution structure of the integral human membrane protein VDAC-1 in detergent micelles. Science 321:1206–1210

    Article  ADS  Google Scholar 

  • Hino M, Kataoka M, Kajimoto K, Yamamoto T, Kido J, Shinohara Y, Baba Y (2008) Efficiency of cell-free protein synthesis based on a crude cell extract from Escherichia coli, wheat germ, and rabbit reticulocytes. J Biotechnol 133:183–189

    Article  Google Scholar 

  • Hirao I, Ohtsuki T, Fujiwara T, Mitsui T, Yokogawa T, Okuni T, Nakayama H, Takio K, Yabuki T, Kigawa T, Kodama K, Yokogawa T, Nishikawa K, Yokoyama S (2002) An unnatural base pair for incorporating amino acid analogs into proteins. Nat Biotechnol 20:177–182

    Article  Google Scholar 

  • Hovijitra NT, Wuu JJ, Peaker B, Swartz JR (2009) Cell-free synthesis of functional aquaporin Z in synthetic liposomes. Biotechnol Bioeng 104:40–49

    Article  Google Scholar 

  • Hwang PM, Choy WY, Lo EI, Chen L, Forman-Kay JD, Raetz CR, Prive GG, Bishop RE, Kay LE (2002) Solution structure and dynamics of the outer membrane enzyme PagP by NMR. Proc Natl Acad Sci USA 99:13560–13565

    Article  ADS  Google Scholar 

  • 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

    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 

  • Jewett MC, Swartz JR (2004a) Rapid expression and purification of 100 nmol quantities of active protein using cell-free protein synthesis. Biotechnol Prog 20:102–109

    Article  Google Scholar 

  • Jewett MC, Swartz JR (2004b) Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm. Biotechnol Bioeng 87:465–472

    Article  Google Scholar 

  • Jewett MC, Swartz JR (2004c) Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnol Bioeng 86:19–26

    Article  Google Scholar 

  • Jia X, Ozawa K, Loscha K, Otting G (2009) Glutarate and N-acetyl-L-glutamate buffers for cell-free synthesis of selectively (15)N-labelled proteins. J Biomol NMR 44:59–67

    Article  Google Scholar 

  • Johansson MU, Alioth S, Hu K, Walser R, Koebnik R, Pervushin K (2007) A minimal transmembrane beta-barrel platform protein studied by nuclear magnetic resonance. Biochemistry 46:1128–1140

    Article  Google Scholar 

  • Kainosho M, Tsuji T (1982) Assignment of the three methionyl carbonyl carbon resonances in Streptomyces subtilisin inhibitor by a carbon-13 and nitrogen-15 double-labeling technique. A new strategy for structural studies of proteins in solution. Biochemistry 21:6273–6279

    Article  Google Scholar 

  • Kainosho M, Torizawa T, Iwashita Y, Terauchi T, Mei Ono A, Guntert P (2006) Optimal isotope labelling for NMR protein structure determinations. Nature 440:52–57

    Article  ADS  Google Scholar 

  • 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

    Article  Google Scholar 

  • Kamonchanok S, Balog CI, van der Does AM, Booth R, de Grip WJ, Deelder AM, Bakker RA, Leurs R, Hensbergen PJ (2008) GPCR proteomics: mass spectrometric and functional analysis of histamine H1 receptor after baculovirus-driven and in vitro cell free expression. J Proteome Res 7:621–629

    Article  Google Scholar 

  • Keller T, Schwarz D, Bernhard F, Dotsch V, Hunte C, Gorboulev V, Koepsell H (2008) Cell free expression and functional reconstitution of eukaryotic drug transporters. Biochemistry 47:4552–4564

    Article  Google Scholar 

  • Kigawa T, Yokoyama S (1991) A continuous cell-free protein synthesis system for coupled transcription-translation. J Biochem 110:166–168

    Google Scholar 

  • Kigawa T, Muto Y, Yokoyama S (1995) Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis. J Biomol NMR 6:129–134

    Article  Google Scholar 

  • Kigawa T, Yabuki T, Yoshida Y, Tsutsui M, Ito Y, Shibata T, Yokoyama S (1999) Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett 442:15–19

    Article  Google Scholar 

  • Kim DM, Choi CY (1996) A semicontinuous prokaryotic coupled transcription/translation system using a dialysis membrane. Biotechnol Prog 12:645–649

    Article  Google Scholar 

  • Kim DM, Swartz JR (1999) Prolonging cell-free protein synthesis with a novel ATP regeneration system. Biotechnol Bioeng 66:180–188

    Article  Google Scholar 

  • Kim DM, Swartz JR (2000) Prolonging cell-free protein synthesis by selective reagent additions. Biotechnol Prog 16:385–390

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • Kopeina GS, Afonina ZA, Gromova KV, Shirokov VA, Vasiliev VD, Spirin AS (2008) Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA. Nucleic Acids Res 36:2476–2488

    Article  Google Scholar 

  • Lee L, Sykes BD (1980) Strategies for the uses of lanthanide NMR shift probes in the determination of protein structure in solutio. Application to the EF calcium binding site of carp parvalbumin. Biophys J 32:193–210

    Article  Google Scholar 

  • Leitz AJ, Bayburt TH, Barnakov AN, Springer BA, Sligar SG (2006) Functional reconstitution of Beta2-adrenergic receptors utilizing self-assembling Nanodisc technology. Biotechniques 40:601–602, 604, 606, passim

    Google Scholar 

  • Liang B, Tamm LK (2007) Structure of outer membrane protein G by solution NMR spectroscopy. Proc Natl Acad Sci USA 104:16140–16145

    Article  ADS  Google Scholar 

  • Liu XM, Sonar S, Lee CP, Coleman M, RajBhandary UL, Rothschild KJ (1995) Site-directed isotope labeling and FTIR spectroscopy: assignment of tyrosine bands in the bR→M difference spectrum of bacteriorhodopsin. Biophys Chem 56:63–70

    Article  Google Scholar 

  • Liu DV, Zawada JF, Swartz JR (2005) Streamlining Escherichia coli S30 extract preparation for economical cell-free protein synthesis. Biotechnol Prog 21:460–465

    Article  MATH  Google Scholar 

  • Matsuda T, Koshiba S, Tochio N, Seki E, Iwasaki N, Yabuki T, Inoue M, Yokoyama S, Kigawa T (2007) Improving cell-free protein synthesis for stable-isotope labeling. J Biomol NMR 37:225–229

    Article  Google Scholar 

  • McIntosh LP, Dahlquist FW (1990) Biosynthetic incorporation of 15N and 13C for assignment and interpretation of nuclear magnetic resonance spectra of proteins. Q Rev Biophys 23:1–38

    Article  Google Scholar 

  • Morgner N, Barth HD, Brutschy B, Scheffer U, Breitung S, Gobel M (2008) Binding sites of the viral RNA element TAR and of TAR mutants for various peptide ligands, probed with LILBID: a new laser mass spectrometry. J Am Soc Mass Spectrom 19:1600–1611

    Article  Google Scholar 

  • Morita EH, Shimizu M, Ogasawara T, Endo Y, Tanaka R, Kohno T (2004) A novel way of amino acid-specific assignment in (1)H-(15)N HSQC spectra with a wheat germ cell-free protein synthesis system. J Biomol NMR 30:37–45

    Article  Google Scholar 

  • Muranaka N, Miura M, Taira H, Hohsaka T (2007) Incorporation of unnatural non-alpha-amino acids into the N terminus of proteins in a cell-free translation system. Chembiochem 8:1650–1653

    Article  Google Scholar 

  • Nirenberg MW (1963) Cell-free protein synthesis directed by messenger RNA. Methods Enzymol 6:17–23

    Article  Google Scholar 

  • Noren CJ, Anthony-Cahill SJ, Griffith MC, Schultz PG (1989) A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244:182–188

    Article  ADS  Google Scholar 

  • Nozawa A, Nanamiya H, Miyata T, Linka N, Endo Y, Weber AP, Tozawa Y (2007) A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters. Plant Cell Physiol 48:1815–1820

    Article  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 

  • Ozawa K, Dixon NE, Otting G (2005) Cell-free synthesis of 15N-labeled proteins for NMR studies. IUBMB Life 57:615–622

    Article  Google Scholar 

  • Ozawa K, Wu PS, Dixon NE, Otting G (2006) N-Labelled proteins by cell-free protein synthesis. Strategies for high-throughput NMR studies of proteins and protein-ligand complexes. FEBS J 273:4154–4159

    Article  Google Scholar 

  • Parker MJ, Aulton-Jones M, Hounslow AM, Craven CJ (2004) A combinatorial selective labeling method for the assignment of backbone amide NMR resonances. J Am Chem Soc 126:5020–5021

    Article  Google Scholar 

  • Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371

    Article  ADS  Google Scholar 

  • Poget SF, Cahill SM, Girvin ME (2007) Isotropic bicelles stabilize the functional form of a small multidrug-resistance pump for NMR structural studies. J Am Chem Soc 129:2432–2433

    Article  Google Scholar 

  • Prosser RS, Evanics F, Kitevski JL, Al-Abdul-Wahid MS (2006) Current applications of bicelles in NMR studies of membrane-associated amphiphiles and proteins. Biochemistry 45:8453–8465

    Article  Google Scholar 

  • Reckel S, Sobhanifar S, Schneider B, Junge F, Schwarz D, Durst F, Lohr F, Guntert P, Bernhard F, Dotsch V (2008) Transmembrane segment enhanced labeling as a tool for the backbone assignment of alpha-helical membrane proteins. Proc Natl Acad Sci USA 105:8262–8267

    Article  ADS  Google Scholar 

  • Roosild TP, Greenwald J, Vega M, Castronovo S, Riek R, Choe S (2005) NMR structure of Mistic, a membrane-integrating protein for membrane protein expression. Science 307:1317–1321

    Article  ADS  Google Scholar 

  • Sanders CRII, Schwonek JP (1992) Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state NMR. Biochemistry 31:8898–8905

    Article  Google Scholar 

  • Schnell JR, Chou JJ (2008) Structure and mechanism of the M2 proton channel of influenza A virus. Nature 451:591–595

    Article  ADS  Google Scholar 

  • Schwarz D, Junge F, Durst F, Frolich N, Schneider B, Reckel S, Sobhanifar S, Dotsch 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 

  • Senes A, Gerstein M, Engelman DM (2000) Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with beta-branched residues at neighboring positions. J Mol Biol 296:921–936

    Article  Google Scholar 

  • Shi J, Pelton JG, Cho HS, Wemmer DE (2004) Protein signal assignments using specific labeling and cell-free synthesis. J Biomol NMR 28:235–247

    Article  MATH  Google Scholar 

  • Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T (2001) Cell-free translation reconstituted with purified components. Nat Biotechnol 19:751–755

    Article  Google Scholar 

  • Shimizu Y, Kanamori T, Ueda T (2005) Protein synthesis by pure translation systems. Methods 36:299–304

    Article  Google Scholar 

  • Sonar S, Lee CP, Coleman M, Patel N, Liu X, Marti T, Khorana HG, RajBhandary UL, Rothschild KJ (1994) Site-directed isotope labelling and FTIR spectroscopy of bacteriorhodopsin. Nat Struct Biol 1:512–517

    Article  Google Scholar 

  • Spirin AS, Baranov VI, Ryabova LA, Ovodov SY, Alakhov YB (1988) A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242:1162–1164

    Article  ADS  Google Scholar 

  • Swartz JR, Jewett MC, Woodrow KA (2004) Cell-free protein synthesis with prokaryotic combined transcription-translation. Methods Mol Biol 267:169–182

    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 

  • Trbovic N, Klammt C, Koglin A, Lohr F, Bernhard F, Dotsch V (2005) Efficient strategy for the rapid backbone assignment of membrane proteins. J Am Chem Soc 127:13504–13505

    Article  Google Scholar 

  • Van Horn WD, Kim HJ, Ellis CD, Hadziselimovic A, Sulistijo ES, Karra MD, Tian C, Sonnichsen FD, Sanders CR (2009) Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase. Science 324:1726–1729

    Article  ADS  Google Scholar 

  • Veglia G, Opella SJ (2000) Lanthanide ion binding to adventitious sites aligns membrane proteins in micelles for solution NMR spectroscopy. J Am Chem Soc 122:11733–11734

    Article  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 

  • Vold RR, Prosser RS, Deese AJ (1997) Isotropic solutions of phospholipid bicelles: a new membrane mimetic for high-resolution NMR studies of polypeptides. J Biomol NMR 9:329–335

    Article  Google Scholar 

  • Weigelt J, van Dongen M, Uppenberg J, Schultz J, Wikstrom M (2002) Site-selective screening by NMR spectroscopy with labeled amino acid pairs. J Am Chem Soc 124:2446–2447

    Article  Google Scholar 

  • Whorton MR, Bokoch MP, Rasmussen SG, Huang B, Zare RN, Kobilka B, Sunahara RK (2007) A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc Natl Acad Sci USA 104:7682–7687

    Article  ADS  Google Scholar 

  • Wu PS, Ozawa K, Jergic S, Su XC, Dixon NE, Otting G (2006) Amino-acid type identification in 15N-HSQC spectra by combinatorial selective 15N-labelling. J Biomol NMR 34:13–21

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Yabuki T, Kigawa T, Dohmae N, Takio K, Terada T, Ito Y, Laue ED, Cooper JA, Kainosho M, Yokoyama S (1998) Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis. J Biomol NMR 11:295–306

    Article  Google Scholar 

  • Zhou Y, Cierpicki T, Jimenez RH, Lukasik SM, Ellena JF, Cafiso DS, Kadokura H, Beckwith J, Bushweller JH (2008) NMR solution structure of the integral membrane enzyme DsbB: functional insights into DsbB-catalyzed disulfide bond formation. Mol Cell 31:896–908

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance (BMRZ), Goethe University, Frankfurt/Main, Germany

    Solmaz Sobhanifar, Sina Reckel, Friederike Junge, Daniel Schwarz, Lei Kai, Mikhail Karbyshev, Frank Löhr, Frank Bernhard & Volker Dötsch

  2. Cluster of Excellence Frankfurt (Macromolecular Complexes), Goethe University, Frankfurt/Main, Germany

    Solmaz Sobhanifar, Sina Reckel, Friederike Junge, Daniel Schwarz, Lei Kai, Mikhail Karbyshev, Frank Löhr, Frank Bernhard & Volker Dötsch

Authors
  1. Solmaz Sobhanifar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sina Reckel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Friederike Junge
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel Schwarz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lei Kai
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mikhail Karbyshev
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Frank Löhr
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Frank Bernhard
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Volker Dötsch
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Volker Dötsch.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sobhanifar, S., Reckel, S., Junge, F. et al. Cell-free expression and stable isotope labelling strategies for membrane proteins. J Biomol NMR 46, 33–43 (2010). https://doi.org/10.1007/s10858-009-9364-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-009-9364-5

Keywords

Search

Navigation