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FRENCH INFRASTRUCTURE FOR INTEGRATED STRUCTURAL BIOLOGY

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INSTRUCT CENTRE - FRANCE 2 - ISBG

LIBRARY BASED CONSTRUCT SCREENING

ESPRIT: Screening of tens of thousands of constructs of a single gene to identify well-behaving soluble constructs

The ESPRIT technology has been developed in the Hart lab at EMBL to express proteins whose domain boundaries are difficult to predict. It does not aim to replace the initial PCR cloning experiments based upon careful inspection of the protein sequence, but provides a rescue strategy when this fails - as it often does. ESPRIT, which stands for “expression of soluble proteins by random incremental truncation”, is a directed evolution-type process combining random deletion mutagenesis with high throughput solubility screening. We use exonuclease to truncate the ends of the target gene sequence in a sequential manner, thereby generating all possible construct termini for downstream testing. Up to 28,000 constructs per gene are isolated using colony picking robots and gridded out to form high density colony arrays for protein expression testing. For detection of soluble constructs in the library, the efficiency of in vivo biotinylation of a fused C-ter peptide is measured using fluorescent probes. Recent developments include adaptation to protein complexes using a coexpression system (An et al., 2011a) and incorporation of a genetic selection to eliminate out-of-frame constructs from the library, a step that greatly enhances the screening power of ESPRIT and its application to more challenging systems (An et al., 2011b).

See more at:

https://www.structuralbiology.eu/update/platform/esprit-library-based-construct-screening-for-soluble-expression-in-grenoble-france/