Abstract
Protein perdeuteration approaches have tremendous value in protein NMR studies, but are limited by the high cost of perdeuterated media. Here, we demonstrate that E. coli cultures expressing proteins using either the condensed single protein production method (cSPP), or conventional pET expression plasmids, can be condensed prior to protein expression, thereby providing high-quality 2H, 13C, 15N-enriched protein samples at 2.5–10% the cost of traditional methods. As an example of the value of such inexpensively-produced perdeuterated proteins, we produced 2H, 13C, 15N-enriched E. coli cold shock protein A (CspA) and EnvZb in 40× condensed phase media, and obtained NMR spectra suitable for 3D structure determination. The cSPP system was also used to produce 2H, 13C, 15N-enriched E. coli plasma membrane protein YaiZ and outer membrane protein X (OmpX) in condensed phase. NMR spectra can be obtained for these membrane proteins produced in the cSPP system following simple detergent extraction, without extensive purification or reconstitution. This allows a membrane protein’s structural and functional properties to be characterized prior to reconstitution, or as a probe of the effects of subsequent purification steps on the structural integrity of membrane proteins. We also provide a standardized protocol for production of perdeuterated proteins using the cSPP system. The 10–40 fold reduction in costs of fermentation media provided by using a condensed culture system opens the door to many new applications for perdeuterated proteins in spectroscopic and crystallographic studies.
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References
Arora A, Abildgraad F, Bushweller JH, Tamm LK (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat Struct Biol 8:334–338
Crespi HL, Katz JJ (1969) High resolution proton magnetic resonance studies of fully deuterated and isotope hybrid proteins. Nature 224:560–562
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
Fernandez C, Adeishvili K, Wuthrich K (2001) Transverse relaxation-optimized NR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine miscelles. Proc Natl Acad Sci USA 98:2358–2363
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
Filip C, Fletcher G, Wulff JL, Earhart CF (1973) Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sacrosinate. J Bacteriol 115:717–722
Gardner KH, Kay LE (1997) Production and incorporation of 15N, 13C, 2H (1H-δ1 Methyl) isoleucine into proteins for multidimensional NMR studies. J Am Chem Soc 119:7599–7600
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
Goddard TD, Kneller DG (2008) SPARKY 3. University of California, San Francisco
Goto NK, Gardner KH, Mueller GA, Willis RC, Kay LE (1999) A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins. J Biomol NMR 13:369–374
Grzesiek S, Anglister J, Ren H, Bax A (1993) 13C line narrowing by 2H decoupling in 2H/13C/15N enriched proteins. Application to triple resonance 4D J connectivity of sequential amides. J Am Chem Soc 115:4369–4370
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
Huang YJ, Hang D, Lu LJ, Tong L, Gerstein MB, Montelione GT (2008) Targeting the human cancer pathway protein interaction network by structural genomics. Mol Cell Proteomics 7:2048–2060
Jansson M, Li Y-C, Jendeberg L, Anderson S, Montelione GT, Nilsson B (1996) High-level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli. J Biomol NMR 7:131–141
Kossiakoff AA, Spencer SA (1981) Direct determination of the protonation states of aspartic acid-102 and histidine-57 in the tetrahedral intermediate of the seriene proteases: neutron structure of trypsin. Biochemistry 20:6462–6474
LeMaster DM, Richards FM (1988) NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteration. Biochemistry 27:142–150
Markley JL, Putter I, Jardetzky O (1968) High-resolution nuclear magnetic resonance spectra of selectively deuterated staphylococcal nuclease. Science 161:1249–1251
Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20:71–75
Moseley HN, Monleon D, Montelione GT (2001) Automatic determination of protein backbone resonance assignments from triple resonance nuclear magnetic resonance data. Methods Enzymol 339:91–108
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 avenua to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371
Rajesh S, Neitlispach D, Nakayama H, Takio K, Laue ED, Shibata T, Ito Y (2003) A novel method for the biosynthesis of deuterated proteins with selective protonation at the aromatic rings of Phe, Tyr, and Trp. J Biomol NMR 27:81–86
Rosen MK, Gardner KH, Willis RC, Parris WE, Pawson T, Kay LE (1996) Selective methyl group protonation of perdeuterated proteins. J Mol Biol 263:627–636
Schneider WM, Inouye M, Montelione GT, Roth MJ (2009) Independently inducible system of gene expression for condensed single protein production (cSPP) suitable for high efficiency isotope enrichment. J Struct Funct Genomics 10:219–225
Sivashanmugam A, Murray V, Cui C, Zhang Y, Wang J, Li Q (2009) Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci 18:936–948
Suzuki M, Mao L, Inouye M (2007) Single protein production (SPP) system in Escherichia coli. Nat Protoc 2:1802–1810
Suzuki M, Rohini R, Zheng H, Woychik N, Inouye M (2006) Bacterial bioreactors for high yield production of recombinant protein. J Biol Chem 281:37559–37565
Suzuki M, Zhang J, Liu M, Woychik NA, Inouye M (2005) Single protein production in living cells facilitated by an mRNA interferase. Mol Cell 18:253–261
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 5:339–344
Venters RA, Metzler WJ, Spicer LD, Mueller L, Farmer BT II (1995) Use of 1HN-1HN NOEs to determine protein global folds in perdeuterated proteins. J Am Chem Soc 117:9592–9593
Zheng D, Huang YJ, Moseley HNB, Xiao R, Aramini J, Swapna GVT, Montelione GT (2003) Automated protein fold determination using a minimal NMR constraint strategy. Protein Sci 12:1232–1246
Acknowledgments
We thank Drs. Rajeswari Mani and Rong Xiao for helpful discussions and comments on the manuscript. This work was supported by the National Institutes of General Medical Science Protein Structure Initiative program, grants U54 GM074958 (to G.T.M and M.I.) and U54 GM75026 (G.T.M. and M.I.), and by grant RO1 GM070837 (to M.J.R.). W.M.S. was supported by NIH training grants T32 GM08360 and T32 A1007403.
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William M. Schneider, Yuefeng Tang, S. Thangminlal Vaiphei, and Lili Mao contributed equally to this work.
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Schneider, W.M., Tang, Y., Vaiphei, S.T. et al. Efficient condensed-phase production of perdeuterated soluble and membrane proteins. J Struct Funct Genomics 11, 143–154 (2010). https://doi.org/10.1007/s10969-010-9083-x
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DOI: https://doi.org/10.1007/s10969-010-9083-x