ESPRIT: A Method for Defining Soluble Expression Constructs in Poorly Understood Gene Sequences

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1586)

Abstract

Production of soluble, purifiable domains or multi-domain fragments of proteins is a prerequisite for structural biology and other applications. When target sequences are poorly annotated, or when there are few similar sequences available for alignments, identification of domains can be problematic. A method called expression of soluble proteins by random incremental truncation (ESPRIT) addresses this problem by high-throughput automated screening of tens of thousands of enzymatically truncated gene fragments. Rare soluble constructs are identified by experimental screening, and the boundaries revealed by DNA sequencing.

Key words

Protein expression Random library Directed evolution 

Notes

Acknowledgments

The ESPRIT platform of the Grenoble Instruct Centre (ISBG: UMS 3518 CNRS-CEA-UJF-EMBL) receives current financial support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). Recent developments were funded from the European Community Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement 283570).

References

  1. 1.
    Dosztányi Z, Sandor M, Tompa P et al (2007) Prediction of protein disorder at the domain level. Curr Protein Pept Sci 8:161–171CrossRefPubMedGoogle Scholar
  2. 2.
    Yumerefendi H, Tarendeau F, Mas PJ et al (2010) ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets. J Struct Biol 172:66–74CrossRefPubMedGoogle Scholar
  3. 3.
    Hart DJ, Waldo GS (2013) Library methods for structural biology of challenging proteins and their complexes. Curr Opin Struct Biol 23:403–408CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Yumerefendi H, Desravines DC, Hart DJ (2011) Library-based methods for identification of soluble expression constructs. Methods 55:38–43CrossRefPubMedGoogle Scholar
  5. 5.
    Ostermeier M, Lutz S (2003) The creation of ITCHY hybrid protein libraries. Methods Mol Biol 231:129–141PubMedGoogle Scholar
  6. 6.
    Beckett D, Kovaleva E, Schatz PJ (1999) A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci 8:921–929CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    An Y, Meresse P, Mas PJ et al (2011) CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes. PLoS One 6:e16261CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    An Y, Yumerefendi H, Mas PJ et al (2011) ORF-selector ESPRIT: a second generation library screen for soluble protein expression employing precise open reading frame selection. J Struct Biol 175:189–197CrossRefPubMedGoogle Scholar
  9. 9.
    Tarendeau F, Boudet J, Guilligay D et al (2007) Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit. Nat Struct Mol Biol 14:229–233CrossRefPubMedGoogle Scholar
  10. 10.
    Tarendeau F, Crepin T, Guilligay D et al (2008) Host determinant residue lysine 627 lies on the surface of a discrete, folded domain of influenza virus polymerase PB2 subunit. PLoS Pathog 4:e1000136CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Guilligay D, Tarendeau F, Resa-Infante P et al (2008) The structural basis for cap binding by influenza virus polymerase subunit PB2. Nat Struct Mol Biol 15:500–506CrossRefPubMedGoogle Scholar
  12. 12.
    Nadal M, Mas PJ, Blanco AG et al (2010) Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain. Proc Natl Acad Sci U S A 107:16078–16083CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Angelini A, Tosi T, Mas P et al (2009) Expression of Helicobacter pylori CagA domains by library-based construct screening. FEBS J 276:816–824CrossRefPubMedGoogle Scholar
  14. 14.
    Rawlings AE, Levdikov VM, Blagova E et al (2010) Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen. Protein Eng Des Sel 23:817–825CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Büssow K, Cahill D, Nietfeld W et al (1998) A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library. Nucleic Acids Res 26:5007–5008CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Integrated Structural Biology Grenoble (ISBG)CNRS, CEA, Université Grenoble Alpes, EMBLGrenobleFrance
  2. 2.Institut de Biologie Structurale (IBS)CNRS, CEA, Université Grenoble AlpesGrenobleFrance

Personalised recommendations