Library Methods

Library methods for soluble expression of challenging proteins

Darren Hart Darren Hart Institut de Biologie Structurale email hidden

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Structural biologists often work on proteins that lack accurate domain annotations. When the full-length protein cannot be expressed and a domain-focused approach is necessary, problems arise since it is unclear how to design high yielding, soluble expression constructs. Some proteins have little or no sequence similarity to others and this prevents domain identification using multiple sequence alignments. More often, some functional annotation exists e.g. from mutagenesis or deletion studies, but these regions do not define well the structural boundaries. Even when a soluble construct is obtained, disordered extensions may confound crystallisation attempts.

Using DNA deletion or fragmentation protocols, libraries of tens of thousands of constructs of a single gene are synthesised and screened for rare clones that produce folded soluble domains. These are identified empirically and require no knowledge of the domain content of the target. The systems here can address single proteins, or simple complexes, and can be used to identify domains de novo, or to improve existing domains for crystallisation.

For a review of this subject, please see:Yumerefendi H, Desravines DC, Hart DJ. Library-based methods for identification of soluble expression constructs. Methods. 2011 Jun 23. [Epub ahead of print]

PMID: 21723393Abstract When expression or crystallisation of a protein target in its wild-type full-length form proves problematic, a common strategy is to divide it into subconstructs comprising one or more domains. Rational construct design is not always successful, especially with targets for which there are few similar sequences to generate multiple sequence alignments. Even when this is possible, expression constructs may still fail to yield soluble protein, commonly expressing insolubly or at unusable yields. To address this, several new methods have been described that borrow concepts from the field of directed evolution whereby a random library is generated encompassing construct diversity; this is then screened to identify soluble constructs empirically. Here, we review progress in this area.

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