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In Vitro Production of Perdeuterated Proteins in H2O for Biomolecular NMR Studies

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Structural Genomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2199))

Abstract

The cell-free synthesis is an efficient strategy to produce in large scale protein samples for structural investigations. In vitro synthesis allows for significant reduction of production time, simplification of purification steps and enables production of both soluble and membrane proteins. The cell-free reaction is an open system and can be performed in presence of many additives such as cofactors, inhibitors, redox systems, chaperones, detergents, lipids, nanodisks, and surfactants to allow for the expression of toxic membrane proteins or intrinsically disordered proteins. In this chapter we present protocols to prepare E. coli S30 cellular extracts, T7 RNA polymerase, and their use for in vitro protein expression. Optimizations of the protocol are presented for preparation of protein samples enriched in deuterium, a prerequisite for the study of high-molecular-weight proteins by NMR spectroscopy. An efficient production of perdeuterated proteins is achieved together with a full protonation of all the amide NMR probes, without suffering from residual protonation on aliphatic carbons. Application to the production of the 468 kDa TET2 protein assembly for NMR investigations is presented.

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References

  1. Wüthrich K (1986) NMR of proteins and nucleic acids. Wiley, New York

    Book  Google Scholar 

  2. Bax A (1989) Two-dimensional NMR and protein structure. Annu Rev Biochem 58:223–256

    Article  CAS  Google Scholar 

  3. Ikura M, Kay LE, Bax A (1990) A novel approach for sequential assignment of 1H, 13C, and 15N spectra of larger proteins: heteronuclear triple-resonance NMR spectroscopy. Application to calmodulin. Biochemistry 29:4659–4667

    Article  CAS  Google Scholar 

  4. Kay LE, Ikura M, Tschudin R, Bax A (1990) Three-dimensional triple resonance NMR spectroscopy of isotopically enriched proteins. J Magn Reson 213:442–445

    Google Scholar 

  5. Bax A (2011) Triple resonance three-dimensional protein NMR: before it became a black box. J Magn Reson 89:496–514

    Google Scholar 

  6. Ikura M, Clore GM, Gronenborn AM, Zhu G, Klee CB, Bax A (1992) Solution structure of a calmodulin-target peptide complex by multi-dimensional NMR. Science 256:632–638

    Article  CAS  Google Scholar 

  7. Yamazaki T, Lee W, Arrowsmith CH, Muhandiram DR, Kay LE (1994) A suite of triple resonance NMR experiments for the backbone assignment of 15N, 13C, 2H labeled proteins with high sensitivity. J Am Chem Soc 116:11655–11666. (Article)

    Article  CAS  Google Scholar 

  8. Venters RA, Huang C-C, Farmer BT II, Trolard R, Spicer LD, Fierke CA (1995) High-level 2H/13C/15N labeling of proteins for NMR studies. J Biomol NMR 5:339–244

    Article  CAS  Google Scholar 

  9. Muchmore SW, Sattler M, Liang H, Meadows RP, Harlan JE, Yoon HS, Nettesheim D, Chang BS, Thompson CB, Wong SL, Ng SL, Fesik SW (1996) X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature 381:335–341

    Article  CAS  Google Scholar 

  10. 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  CAS  Google Scholar 

  11. Pervushin K, Riek R, Wider G, Wüthrich 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 U S A 94:12366–12371

    Article  CAS  Google Scholar 

  12. Riek R, Wider G, Pervushin K, Wüthrich K (1999) Polarization transfer by cross-correlated relaxation in solution NMR with very large molecules. Proc Natl Acad Sci U S A 96:4918–4923

    Article  CAS  Google Scholar 

  13. Tugarinov V, Muhandiram R, Ayed A, Kay LE (2002) Four-dimensional NMR spectroscopy of a 723-residue protein: chemical shift assignments and secondary structure of malate synthase g. J Am Chem Soc 124:10025–10035

    Article  CAS  Google Scholar 

  14. Wider G, Wüthrich K (1999) NMR spectroscopy of large molecules and multimolecular assemblies in solution. Curr Opin Struct Biol 9:594–601

    Article  CAS  Google Scholar 

  15. Fiaux J, Bertelsen EB, Horwich AL, Wüthrich K (2002) NMR analysis of a 900K GroEL GroES complex. Nature 418:207–211

    Article  CAS  Google Scholar 

  16. Sounier R, Blanchard L, Wu Z, Boisbouvier J (2007) High-accuracy distance measurement between remote Methyls in specifically protonated proteins. J Am Chem Soc 129:472–473

    Article  CAS  Google Scholar 

  17. Fiaux J, Bertelsen EB, Horwich AL, Wüthrich K (2004) Uniform and residue-specific 15N-labeling of proteins on a highly deuterated background. J Biomol NMR 29:289–297

    Article  CAS  Google Scholar 

  18. Rasia R, Noirclerc-Savoye M, Gallet B, Bologna N, Plevin M, Blanchard L, Palatnik J, Brutscher B, Vernet T, Boisbouvier J (2009) Parallel screening and optimization of protein constructs for structural studies. Protein Sci 18:434–439

    Article  CAS  Google Scholar 

  19. Smith BO, Ito Y, Raine A, Teichmann S, Ben-Tovim L et al (1996) An approach to structure determination using limited NMR data from larger proteins selectively protonated at specific residue types. J Biomol NMR 8:360–368

    Article  CAS  Google Scholar 

  20. Zubay G (1973) In vitro synthesis of protein in microbial systems. Ann Rev Genet 7:267–287

    Article  CAS  Google Scholar 

  21. Pratt C (1980) Kinetics and regulation of cell-free alkaline phosphatase synthesis. J Bacteriol 143(3):1265–1274

    Article  CAS  Google Scholar 

  22. Roberts BE, Paterson BM (1973) Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ. PNAS 70:2330–2334

    Article  CAS  Google Scholar 

  23. Klammt C, Löhr F, Schafer B, Haase W, Dötsch 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. https://doi.org/10.1111/j.1432-1033.2003.03959.x

    Article  CAS  Google Scholar 

  24. Davanloo P, Rosenberg AH, Dunn JJ, Studier FW (1984) Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 81:2035–2039

    Article  CAS  Google Scholar 

  25. Zawadzki V, Gross HJ (1991) Rapid and simple purification of T7 RNA polymerase. Nucleic Acids Res 25:1948

    Article  Google Scholar 

  26. 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  CAS  Google Scholar 

  27. 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  CAS  Google Scholar 

  28. Martin GA, Kawaguchi R, Lam Y, DeGiovanni A, Fukushima M, Mutter W (2001) High-yield, in vitro protein expression using a continuous-exchange, coupled transcription/translation system. BioTechniques 31:948–953

    Article  CAS  Google Scholar 

  29. Haberstock S, Roos C, Hoevels Y, Dötsch V, Schnapp G, Pautsch A, Bernhard F (2012) A systematic approach to increase the efficiency of membrane protein production in cell-free expression systems. Protein Expr Purif 82:308–316

    Article  CAS  Google Scholar 

  30. 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  CAS  Google Scholar 

  31. Su XC, Loh CT, Qi R, Otting G (2011) Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enzymes for selective 15N-labelling and production of perdeuterated proteins in H2O. J Biomol NMR 50:35–42

    Article  CAS  Google Scholar 

  32. Yokoyama J, Matsuda T, Koshiba S, Tochio N, Kigawa T (2011) A practical method for cell-free protein synthesis to avoid stable isotope scrambling and dilution. Anal Biochem 411:223–229. https://doi.org/10.1016/j.ab.2011.01.017

    Article  CAS  Google Scholar 

  33. Mayerhofer H, Sautron E, Rolland N, Catty P, Seigneurin-Berny D, Pebay-Peyroula E, Ravaud S (2016) Structural insights into the nucleotide-binding domains of the P1B-type ATPases HMA6 and HMA8 from Arabidopsis thaliana. PLoS One 11(11):e0165666. https://doi.org/10.1371/journal.pone.0165666

    Article  CAS  Google Scholar 

  34. Lescop E, Schanda P, Brutscher B (2007) A set of BEST triple-resonance experiments for time-optimized protein resonance assignment. J Magn Reson 187:163–169

    Article  CAS  Google Scholar 

  35. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293

    Article  CAS  Google Scholar 

  36. Sprangers R, Kay LE (2007) Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 445:618–622

    Article  CAS  Google Scholar 

  37. Macek P, Kerfah R, Boeri Erba E, Crublet E, Moriscot C, Schoehn G, Amero C, Boisbouvier J (2017) Unraveling self-assembly pathways of the 468 kDa Proteolytic machine TET2. Sci Adv 3(4):e1601601. https://doi.org/10.1126/sciadv.1601601

    Article  CAS  Google Scholar 

  38. Mas G, Guan J-Y, Crublet E, Colas Debled E, Moriscot C, Gans P, Schoehn G, Macek P, Schanda P, Boisbouvier J (2018) Structural investigation of a chaperonin in action reveals how nucleotide binding regulates the functional cycle. Sci Adv 4(9):eaau4196. https://doi.org/10.1126/sciadv.aau4196

    Article  CAS  Google Scholar 

  39. Franzetti B, Schoehn G, Hernandez JF, Jaquinod M, Ruigrok RW, Zaccai G (2002) Tetrahedral aminopeptidase: a novel large protease complex from archaea. EMBO J 21:2132–2138

    Article  CAS  Google Scholar 

  40. Borissenko L, Groll M (2005) Crystal structure of TET protease reveals complementary protein degradation pathways in prokaryotes. J Mol Biol 346:1207–1219

    Article  CAS  Google Scholar 

  41. Fraga H, Arnaud CA, Gauto DF, Audin MJC, Kurauskas V, Macek P, Krichel C, Guan JY, Boisbouvier J, Sprangers R, Breyton C, Schanda P (2017) Solid-state NMR H-N-(C)-H and H-N-C-C 3D/4D correlation experiments for resonance assignment of large proteins. Chem Phys Chem 18:2697–2703

    Article  CAS  Google Scholar 

  42. Gauto D, Estrozi L, Schwieters C, Effantin G, Macek P, Sounier R, Sivertsen AC, Schmidt E, Kerfah R, Mas G, Colletier JP, Güntert P, Favier A, Schoehn G, Schanda P, Boisbouvier J (2019) Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex. Nat Commun 10(1):1234567890. https://doi.org/10.1038/s41467-019-10490-9

    Article  CAS  Google Scholar 

  43. Bertini I, Luchinat C, Parigi G, Ravera E, Reif B, Turano P (2011) Solid-state NMR of proteins sedimented by ultracentrifugation. Proc Natl Acad Sci U S A 108:10396–10399. https://doi.org/10.1073/pnas.1103854108

    Article  Google Scholar 

  44. 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  CAS  Google Scholar 

  45. Apponyi MA, Ozawa K, Dixon NE, Otting G (2008) Cell-free protein synthesis for analysis by NMR spectroscopy. Methods Mol Biol 426:257–268. https://doi.org/10.1007/978-1-60327-058-8_16

    Article  CAS  Google Scholar 

  46. 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  CAS  Google Scholar 

  47. Pedersen A, Hellberg K, Enberg J, Karlson G (2011) Rational improvement of cell-free protein synthesis. New Biotechnol 28:218–224. https://doi.org/10.1016/j.nbt.2010.06.015

    Article  CAS  Google Scholar 

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Acknowledgments

The authors thank Prof. Eva Pebay-Peyroula for providing the clone of the HMA8 ATPases nucleotide-binding domain from Arabidopsis thaliana. This work used the high field NMR and Cell-Free facilities at the Grenoble Instruct-ERIC Center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB). Platform access was supported by FRISBI (ANR-10-INBS-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). This work was supported by grants from CEA/NMR-Bio (research program C24990) and the Agence Nationale de la Recherche (ANR-17-CE29-0010 CH2-PROBE).

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Author notes

  1. Lionel Imbert and Rachel Lenoir-Capello contributed equally to this work.

Authors and Affiliations

  1. CNRS, CEA, Institut de Biologie Structurale (IBS), University of Grenoble Alpes, Grenoble, France

    Lionel Imbert, Alicia Vallet, Rida Awad, Isabel Ayala, Celine Juillan-Binard, Hubert Mayerhofer, Pierre Gans & Jerome Boisbouvier

  2. CNRS, CEA, EMBL, Integrated Structural Biology Grenoble (ISBG), University of Grenoble Alpes, Grenoble, France

    Lionel Imbert & Celine Juillan-Binard

  3. CNRS, Laboratoire des biomolécules, LBM, Sorbonne Université, École normale supérieure, PSL University, Paris, France

    Rachel Lenoir-Capello & Emeric Miclet

  4. NMR-Bio, Grenoble, France

    Elodie Crublet & Rime Kerfah

Authors
  1. Lionel Imbert
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  2. Rachel Lenoir-Capello
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  6. Isabel Ayala
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  7. Celine Juillan-Binard
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  8. Hubert Mayerhofer
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  9. Rime Kerfah
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  10. Pierre Gans
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  11. Emeric Miclet
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  12. Jerome Boisbouvier
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Correspondence to Jerome Boisbouvier .

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Editors and Affiliations

  1. Department of Applied Biology and Chemical Technology and the State Key Laboratory of Chemical Biology and Drug Discovery, The Hong Kong Polytechnic University, Hung Hom, Hong Kong

    Yu Wai Chen

  2. BMI Biotechnology Appraisals & Investments Limited, Wan Chai, Hong Kong

    Chin-Pang Bennu Yiu

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Imbert, L. et al. (2021). In Vitro Production of Perdeuterated Proteins in H2O for Biomolecular NMR Studies. In: Chen, Y.W., Yiu, CP.B. (eds) Structural Genomics. Methods in Molecular Biology, vol 2199. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0892-0_8

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  • DOI: https://doi.org/10.1007/978-1-0716-0892-0_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0891-3

  • Online ISBN: 978-1-0716-0892-0

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