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J Struct Biol. 2018 Feb;201(2):88-99. doi: 10.1016/j.jsb.2017.08.002. Epub 2017 Aug 16.

Alpha repeat proteins (αRep) as expression and crystallization helpers.

Author information

1
Institute for Integrative Biology of the Cell (I2BC) CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette cedex, France.
2
Institute for Integrative Biology of the Cell (I2BC) CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette cedex, France. Electronic address: philippe.minard@u-psud.fr.

Abstract

We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize.

KEYWORDS:

Artificial repeat proteins; Crystallization helper; Fusion protein; Protein complementation assay; Protein library

PMID:
28823563
DOI:
10.1016/j.jsb.2017.08.002
[Indexed for MEDLINE]

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