Skip to main content
SpringerLink
Log in

Recombinant Intrinsically Disordered Proteins for NMR: Tips and Tricks

  • Chapter
  • First Online:
Intrinsically Disordered Proteins Studied by NMR Spectroscopy

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 870))

Abstract

The growing recognition of the several roles that intrinsically disordered proteins play in biology places an increasing importance on protein sample availability to allow the characterization of their structural and dynamic properties. The sample preparation is therefore the limiting step to allow any biophysical method being able to characterize the properties of an intrinsically disordered protein and to clarify the links between these properties and the associated biological functions.

An increasing array of tools has been recruited to help prepare and characterize the structural and dynamic properties of disordered proteins. This chapter describes their sample preparation, covering the most common drawbacks/barriers usually found working in the laboratory bench. We want this chapter to be the bedside book of any scientist interested in preparing intrinsically disordered protein samples for further biophysical analysis.

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

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Notes

  1. 1.

    http://pedb.vib.be.

  2. 2.

    http://www.dabi.temple.edu/disprot/index.php.

  3. 3.

    http://d2p2.pro.

  4. 4.

    http://www.idpbynmr.eu/home.

  5. 5.

    http://predictioncenter.org.

  6. 6.

    http://bioware.ucd.ie/~compass/biowareweb/Server_pages/slimfinder.php.

  7. 7.

    http://biomine-ws.ece.ualberta.ca/MoRFpred/index.html.

  8. 8.

    http://anchor.enzim.hu.

  9. 9.

    http://www.pdb.org/pdb/home/home.do.

  10. 10.

    http://www.genscript.com/cgi-bin/tools/rare_codon_analysis.

  11. 11.

    http://nihserver.mbi.ucla.edu/RACC/.

  12. 12.

    http://web.expasy.org/ protparam/.

  13. 13.

    http://www.ogic.ca/projects/k2d3/.

References

  • Armstrong DJ, Roman A (1993) The anomalous electrophoretic behavior of the human papillomavirus type 16 E7 protein is due to the high content of acidic amino acid residues. Biochem Biophys Res Commun 192:1380–1387. doi:10.1006/bbrc.1993.1569

    Article  CAS  PubMed  Google Scholar 

  • Arnau J, Arnau J, Lauritzen C et al (2006) Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 48:1–13. doi:10.1016/j.pep.2005.12.002

    Article  CAS  PubMed  Google Scholar 

  • Babich H, Stotzky G (1978) Toxicity of zinc to fungi, bacteria, and coliphages: influence of chloride ions. Appl Environ Microbiol 36:906–914

    PubMed Central  CAS  PubMed  Google Scholar 

  • Bermel W, Bertini I, Felli IC et al (2012) Speeding up sequence specific assignment of IDPs. J Biomol NMR 53:293–301. doi:10.1007/s10858-012-9639-0

    Article  CAS  PubMed  Google Scholar 

  • Bernadó P, Svergun DI (2012a) Structural analysis of intrinsically disordered proteins by small-angle X-ray scattering. Mol BioSyst 8:151–167. doi:10.1039/c1mb05275f

    Article  PubMed  Google Scholar 

  • Bernadó P, Svergun DI (2012b) Analysis of intrinsically disordered proteins by small-angle X-ray scattering. Methods Mol Biol 896:107–122. doi:10.1007/978-1-4614-3704-87

    PubMed  Google Scholar 

  • Bernadó P, Mylonas E, Petoukhov MV et al (2007) Structural characterization of flexible proteins using small-angle X-ray scattering. J Am Chem Soc 129:5656–5664. doi:10.1021/ja069124n

    Article  PubMed  Google Scholar 

  • Block H, Maertens B, Spriestersbach A et al (2009) Immobilized-metal affinity chromatography (IMAC): a review. Meth Enzymol 463:439–473. doi:10.1016/S0076-6879(09)63027-5

    Article  CAS  PubMed  Google Scholar 

  • Brito RMM, Vaz WLC (1986) Determination of the critical micelle concentration of surfactants using the Fluorescent-Probe N-Phenyl-1-Naphthylamine. Anal Biochem 152:250–255. doi:10.1016/0003-2697(86)90406-9

    Article  CAS  PubMed  Google Scholar 

  • Burgess R, Richard R, Murray P (2009) Refolding solubilized inclusion body proteins. Methods Enzymol 463:259–282

    Article  CAS  PubMed  Google Scholar 

  • Coutard B, Danchin EGJ, Oubelaid R et al (2012) Single pH buffer refolding screen for protein from inclusion bodies. Protein Expr Purif 82:352–359. doi:10.1016/j.pep.2012.01.014

    Article  CAS  PubMed  Google Scholar 

  • Cowieson NP, Wensley B, Listwan P et al (2006) An automatable screen for the rapid identification of proteins amenable to refolding. Proteomics 6:1750–1757. doi:10.1002/pmic.200500056

    Article  CAS  PubMed  Google Scholar 

  • Das U, Hariprasad G, Ethayathulla AS et al (2007) Inhibition of protein aggregation: supramolecular assemblies of arginine hold the key. PLoS ONE 2:e1176. doi:10.1371/journal.pone.0001176

    Google Scholar 

  • Davey NE, Haslam NJ, Shields DC et al (2010) SLiMFinder: a web server to find novel, significantly over-represented, short protein motifs. Nucleic Acids Res 38:W534–W539. doi:10.1093/nar/gkq440

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Di Marco VB, Bombi GG (2006) Electrospray mass spectrometry (ESI-MS) in the study of metal–ligand solution equilibria. Mass Spectrom Rev 25:347–379. doi:10.1002/mas.20070

    Article  PubMed  Google Scholar 

  • Disfani FM, Hsu W-L, Mizianty MJ et al (2012) MoRFpred, a computational tool for sequence-based prediction and characterization of short disorder-to-order transitioning binding regions in proteins. Bioinformatics 28:i75–i83. doi:10.1093/bioinformatics/bts209

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Dunker AK, Obradovic Z (2001) The protein trinity—linking function and disorder. Nat Biotechnol 19:805–806. doi:10.1038/nbt0901-805

    Article  CAS  PubMed  Google Scholar 

  • Dunker AK, Lawson JD, Brown CJ et al (2001) Intrinsically disordered protein. J Mol Graph Model 19:26–59

    Article  CAS  PubMed  Google Scholar 

  • Dunker AK, Oldfield CJ, Meng J et al (2008) The unfoldomics decade: an update on intrinsically disordered proteins. BMC Genomics 9(Suppl 2):S1. doi:10.1186/1471-2164-9-S2-S1

    Article  Google Scholar 

  • Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17:353–358. doi:10.1016/j.copbio.2006.06.003

    Article  CAS  PubMed  Google Scholar 

  • Eswar N, Webb B, Marti-Renom MA et al (2006) Comparative protein structure modeling using MODELLER. Curr Protoc Bioinformat UNIT 5.6. doi:10.1002/0471250953.bi0506s15

    Google Scholar 

  • Gasteiger E, Hoogland C, Gattiker A et al (2005) Protein identification and analysis tools on the ExPASy server. 571–607. doi:10.1385/1-59259-890-0:571

    Google Scholar 

  • Getz EB, Xiao M, Chakrabarty T et al (1999) A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal Biochem 273:73–80. doi:10.1006/abio.1999.4203

    Article  CAS  PubMed  Google Scholar 

  • Graceffa P, Jancsó A, Mabuchi K (1992) Modification of acidic residues normalizes sodium dodecyl sulfate-polyacrylamide gel electrophoresis of caldesmon and other proteins that migrate anomalously. Arch Biochem Biophys 297:46–51. doi:10.1016/0003-9861(92)90639-E

    Article  CAS  PubMed  Google Scholar 

  • Gupta BB (1983) Determination of native and denatured milk proteins by high-performance size exclusion chromatography. J Chromatogr A 282:463–475. doi:10.1016/S0021-9673(00)91623-6

    Article  CAS  Google Scholar 

  • Hunt I (2005) From gene to protein: a review of new and enabling technologies for multi-parallel protein expression. Protein Expr Purif 40:1–22. doi:10.1016/j.pep.2004.10.018

    Article  CAS  PubMed  Google Scholar 

  • Ignatova Z, Gierasch LM (2006) Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant. Proc Natl Acad Sci U S A 103:13357–13361. doi:10.1073/pnas.0603772103

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Jacques DA, Guss JM, Svergun DI, Trewhella J (2012) Publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution. Acta Crystallogr D Biol Crystallogr 68:620–626. doi:10.1107/S0907444912012073

    Article  CAS  PubMed  Google Scholar 

  • Jana S, Deb JK (2005) Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 67:289–298. doi:10.1007/s00253-004-1814-0

    Article  CAS  PubMed  Google Scholar 

  • Jumpertz T, Tschapek B, Infed N et al (2011) High-throughput evaluation of the critical micelle concentration of detergents. Anal Biochem 408:64–70. doi:10.1016/j.ab.2010.09.011

    Article  CAS  PubMed  Google Scholar 

  • Kaltashov IA, Bobst CE, Abzalimov RR (2013) Mass spectrometry-based methods to study protein architecture and dynamics. Protein Sci 22:530–544. doi:10.1002/pro.2238

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Katzen F (2007) Gateway ®recombinational cloning: a biological operating system. Expert Opin Drug Discov 2:571–589. doi:10.1517/17460441.2.4.571

    Article  CAS  PubMed  Google Scholar 

  • Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochem. Biophys. Acta (BBA)—Proteins Proteomics 1751:119–139. doi:10.1016/j.bbapap.2005.06.005

    Article  CAS  Google Scholar 

  • Kindermann B, Döring F, Fuchs D et al (2005) Effects of increased cellular zinc levels on gene and protein expression in HT-29 cells. Biometals 18:243–253. doi:10.1007/s10534-005-1247-y

    Article  CAS  PubMed  Google Scholar 

  • Knapman TW, Valette NM, Warriner SL et al (2013) Ion mobility spectrometry-mass spectrometry of intrinsically unfolded proteins: trying to put order into disorder. Curr Anal Chem 9:181–191. doi:10.2174/1573411011309020004

    PubMed Central  CAS  PubMed  Google Scholar 

  • Konarev PV, Petoukhov MV, Volkov VV et al (2006) ATSAS 2.1, a program package for small-angle scattering data analysis. J Appl Crystallogr 39:277–286. doi:10.1107/S0021889806004699

    Article  CAS  Google Scholar 

  • Krezel A, Latajka R, Bujacz GD et al (2003) Coordination properties of tris(2-carboxyethyl)phosphine, a newly introduced thiol reductant, and its oxide. Inorg Chem 42:1994–2003. doi:10.1021/ic025969y

    Article  CAS  PubMed  Google Scholar 

  • Lesley SA (2009) Parallel methods for expression and purification. Methods. Enzymol. 463:767–785

    Google Scholar 

  • Linn S (2009) Strategies and considerations for protein purifications. Methods. Enzymol. 463:9–19

    Google Scholar 

  • Louis-Jeune C, Andrade-Navarro MA, Perez-Iratxeta C (2012) Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. Proteins 80:374–381. doi:10.1002/prot.23188

    Article  CAS  PubMed  Google Scholar 

  • Malhotra A (2009) Tagging for protein expression. In: Methods in Enzymology. Elsevier, pp 239–258

    Google Scholar 

  • Mészáros B, Simon I, Dosztányi Z (2009) Prediction of protein binding regions in disordered proteins. PLoS Comput Biol 5:e1000376. doi:10.1371/journal.pcbi.1000376

    Article  PubMed Central  PubMed  Google Scholar 

  • Oates ME, Romero P, Ishida T et al (2013) D²P²: database of disordered protein predictions. Nucleic Acids Res 41:D508–D516. doi:10.1093/nar/gks1226

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Outten CE, O’Halloran ATV (2001) Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis. Science 292:2488–2492. doi:10.1126/science.1060331

    Article  CAS  PubMed  Google Scholar 

  • Petoukhov MV, Svergun DI (2013) Applications of small-angle X-ray scattering to biomacromolecular solutions. Int J Biochem Cell Biol 45:429–437. doi:10.1016/j.biocel.2012.10.017

    Article  CAS  PubMed  Google Scholar 

  • Petoukhov MV, Franke D, Shkumatov AV et al (2012) New developments in the ATSAS program package for small-angle scattering data analysis. J Appl Crystallogr 45:342–350. doi:10.1107/S0021889812007662

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Qoronfleh MW, Hesterberg LK, Seefeldt MB (2007) Confronting high-throughput protein refolding using high pressure and solution screens. Protein Expr Purif 55:209–224. doi:10.1016/j.pep.2007.05.014

    Article  CAS  PubMed  Google Scholar 

  • Radivojac P, Iakoucheva LM, Oldfield CJ et al (2007) Intrinsic disorder and functional proteomics. Biophys J 92:1439–1456. doi:10.1529/biophysj.106.094045

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Šali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815. doi:10.1006/jmbi.1993.1626

    Article  PubMed  Google Scholar 

  • Schenk PM, Baumann S, Mattes R et al (1995) Improved high-level expression system for eukaryotic genes in Escherichia coli using T7 RNA polymerase and rare ArgtRNAs. Biotechniques 19:196–200

    CAS  PubMed  Google Scholar 

  • Shatzman AR (1995) Expression systems. Curr Opin Biotechnol 6:491–493

    Article  CAS  Google Scholar 

  • Sickmeier M, Hamilton JA, LeGall T et al (2007) DisProt: the database of disordered proteins. Nucleic Acids Res 35:D786–D793. doi:10.1093/nar/gkl893

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Singh SM, Singh SM, Panda AK et al (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99:303–310. doi:10.1263/jbb.99.303

    Article  CAS  PubMed  Google Scholar 

  • Theillet F-X, Kalmar L, Tompa P et al (2013) The alphabet of intrinsic disorder I. Act like a Pro: on the abundance and roles of proline residues in intrinsically disordered proteins. Intrinsically Disord Protein 1:0–12

    Article  Google Scholar 

  • Theillet F-X, Binolfi A, Frembgen-Kesner T et al (2014) Physicochemical properties of cells and their effects on intrinsically disordered proteins (IDPs). Chem Rev 140627063652000. doi:10.1021/cr400695p

    Google Scholar 

  • Tong KI, Yamamoto M, Tanaka T (2008) A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli. J Biomol NMR 42:59–67. doi:10.1007/s10858-008-9264-0

    Article  CAS  PubMed  Google Scholar 

  • Uversky VN (2011) Intrinsically disordered proteins from A to Z. Int J Biochem Cell Biol 43:1090–1103. doi:10.1016/j.biocel.2011.04.001

    Article  CAS  PubMed  Google Scholar 

  • Uversky VN (2013) A decade and a half of protein intrinsic disorder: biology still waits for physics. Protein Sci 22:693–724. doi:10.1002/pro.2261

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Varadi M, Kosol S, Lebrun P et al (2013) pE-DB: a database of structural ensembles of intrinsically disordered and of unfolded proteins. Nucleic Acids Res. doi:10.1093/nar/gkt960

    Google Scholar 

  • Vincentelli R, Canaan S, Campanacci V et al (2004) High-throughput automated refolding screening of inclusion bodies. Protein Sci 13:2782–2792. doi:10.1110/ps.04806004

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ward JJ, Sodhi JS, McGuffin LJ et al (2004) Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. J Mol Biol 337:635–645. doi:10.1016/j.jmb.2004.02.002

    Article  CAS  PubMed  Google Scholar 

  • Weinhandl K, Winkler M, Glieder A et al (2014) Carbon source dependent promoters in yeasts. Microb Cell Fact 13:5. doi:10.1186/1475-2859-13-5

    Article  PubMed Central  PubMed  Google Scholar 

  • Xue B, Dunbrack RL, Williams RW et al (2010) PONDR-FIT: a meta-predictor of intrinsically disordered amino acids. Biochim Biophys Acta 1804:996–1010. doi:10.1016/j.bbapap.2010.01.011

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Kathleen McGreevy and Leonardo Gonnelli are gratefully acknowledged for their comments to the manuscript.

Author information

Authors and Affiliations

  1. Magnetic Resonance Center (CERM), University of Florence, Via Luigi Sacconi 6, 50019, Sesto Fiorentino, Italy

    Eduardo O. Calçada

  2. Giotto Biotech, Via Madonna del Piano 6, 50019, Sesto Fiorentino, Italy

    Magdalena Korsak & Tatiana Kozyreva

Authors
  1. Eduardo O. Calçada
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Magdalena Korsak
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tatiana Kozyreva
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Eduardo O. Calçada .

Editor information

Editors and Affiliations

  1. CERM and Department of Chemistry, "Ugo Schiff" University of Florence, Florence, Italy

    Isabella C. Felli

  2. CERM and Department of Chemistry, "Ugo Schiff" University of Florence, Florence, Italy

    Roberta Pierattelli

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer International Publishing Switzerland

About this chapter

Cite this chapter

Calçada, E., Korsak, M., Kozyreva, T. (2015). Recombinant Intrinsically Disordered Proteins for NMR: Tips and Tricks. In: Felli, I., Pierattelli, R. (eds) Intrinsically Disordered Proteins Studied by NMR Spectroscopy. Advances in Experimental Medicine and Biology, vol 870. Springer, Cham. https://doi.org/10.1007/978-3-319-20164-1_6

Download citation

Publish with us

Policies and ethics

Search

Navigation