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.
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References
Dosztányi Z, Sandor M, Tompa P et al (2007) Prediction of protein disorder at the domain level. Curr Protein Pept Sci 8:161–171
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 172:66–74
Hart DJ, Waldo GS (2013) Library methods for structural biology of challenging proteins and their complexes. Curr Opin Struct Biol 23:403–408
Yumerefendi H, Desravines DC, Hart DJ (2011) Library-based methods for identification of soluble expression constructs. Methods 55:38–43
Ostermeier M, Lutz S (2003) The creation of ITCHY hybrid protein libraries. Methods Mol Biol 231:129–141
Beckett D, Kovaleva E, Schatz PJ (1999) A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci 8:921–929
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:e16261
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 175:189–197
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 14:229–233
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:e1000136
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 15:500–506
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 107:16078–16083
Angelini A, Tosi T, Mas P et al (2009) Expression of Helicobacter pylori CagA domains by library-based construct screening. FEBS J 276:816–824
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 23:817–825
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 26:5007–5008
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).
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Mas, P.J., Hart, D.J. (2017). ESPRIT: A Method for Defining Soluble Expression Constructs in Poorly Understood Gene Sequences. In: Burgess-Brown, N. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 1586. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6887-9_4
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DOI: https://doi.org/10.1007/978-1-4939-6887-9_4
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