Service / Technology Instance

About ESPRIT: Library-Based Screening for Soluble Expression, Grenoble, France

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Expression of Soluble Proteins by Random Incremental Truncation (ESPRIT) is a directed evolution-type process combining random deletion mutagenesis with high throughput solubility screening. 

Service Availability:

Remote

Instruments Available:

User Guide

ESPRIT uses exonuclease to truncate the target gene sequence and generate all possible construct termini. Up to 28,000 clones are isolated using colony picking robots and gridded out to form high density colony arrays for protein expression testing. This means ESPRIT is able to screen of tens of thousands of constructs of a single gene to identify well-behaving soluble constructs.

When the full-length protein cannot be expressed and a domain-focused approach is necessary, it can be difficult to design well-expressing soluble constructs. Common issues are:

  • Some proteins have little or no sequence similarity to others and this prevents domain identification using multiple sequence alignments 
  • Some functional annotation exists e.g. from mutagenesis or deletion studies, but these regions do not define the structural boundaries
  • Even when a soluble construct is obtained, disordered extensions may confound crystallisation attempts

ESPRIT technology was developed to express proteins whose domain boundaries are difficult to predict.

Over the last ten years, the ESPRIT platform has been visited by many European scientists who have brought their problematic targets for screening. About half of these users have returned home with soluble, purified proteins for further study. 

Technology references:

Mas PJ, Hart DJ. (2010) ESPRIT: A Method for Defining Soluble Expression Constructs in Poorly Understood Gene Sequences. Methods Mol Biol. 2017;1586:45-63

Yumerefendi H, et al,. (2010) ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets. J Struct Biol. 2010 Oct;172(1):66-74

An Y, Meresse P, Mas PJ, Hart DJ. (2011) CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes. PLoS One. 2011 Feb 22;6(2):e16261

An Y, et al., (2011) ORF-selector ESPRIT: a second generation library screen for soluble protein expression employing precise open reading frame selection. J Struct Biol. 2011 Aug;175(2):189-97

Hart DJ, Waldo GS, (2013) Library methods for structural biology of challenging proteins and their complexes. Curr Opin Struct Biol. 2013 Jun;23(3):403-8

Examples:

Desravines DC, et al., (2017) Structural Characterization of the SMRT Corepressor Interacting with Histone Deacetylase 7. Sci Rep. 2017 Jun 16;7(1):3678,

Sukackaite R. et al., (2014) Structural and biophysical characterisation of murine rif1 C terminus reveals high specificity for DNA cruciform structures. J Biol Chem. 2014 May 16;289(20):13903-11,

Leupold S, et al., (2017) Structural insights into the architecture of the Shigella flexneri virulence factor IcsA/VirG and motifs involved in polar distribution and secretion. J Struct Biol. 2017 Apr;198(1):19-27

Yerabham ASK, et al., (2017) A structural organization for the Disrupted in Schizophrenia 1 protein, identified by high-throughput screening, reveals distinctly folded regions, which are bisected by mental illness-related mutations. J Biol Chem. 2017 Apr 21;292(16):6468-6477

Rawlings AE, 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. 2010 Nov;23(11):817-25

Nadal M, et al., (2010) Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain. Proc Natl Acad Sci U S A. 2010 Sep 14;107(37):16078-83,

 

Tarbouriech N, et al., (2017) The vaccinia virus DNA polymerase structure provides insights into the mode of processivity factor binding. Nat Commun. 2017 Nov 13;8(1):1455

For information, see the ISBG webpage.

Instruct Centre

Instruct Centre FR2

IBS-ISBG

71 avenue des Martyrs

Grenoble

France

www.ibs.fr & www.isbg.fr

ESPRIT: Library-Based Screening for Soluble Expression, Grenoble, France

Contacts:

Philippe Mas
Philippe Mas
Centre de Biologie Structurale (CBS), CNRS, Univ. Montpellier, Inserm
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Darren Hart
Darren Hart
Centre de Biologie Structurale (CBS), CNRS, Univ. Montpellier, Inserm
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