Institute for Integrative Biology of the Cell (I2BC) CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette cedex, France.
Institute for Integrative Biology of the Cell (I2BC) CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette cedex, France.
J Struct Biol. 2018 Feb;201(2):88-99. doi: 10.1016/j.jsb.2017.08.002. Epub 2017 Aug 16.
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.
我们之前描述了一个基于热稳定 HEAT 样重复序列的高度多样化的人工重复蛋白库，名为αRep。已经选择了专门结合难以结晶的蛋白质的αReps，在几个例子中，它们使得这些蛋白质的结晶成为可能。为了进一步简化生产和结晶实验，我们探索了生产与目标共价结合的嵌合蛋白的方法，其特异性结合剂通过连接子得到加强。虽然具有表达伙伴的嵌合蛋白通常用于增强表达，但这些融合蛋白通常不能用于结晶。但是，有了特定的表达伙伴，如同源的αRep，这就不再是真的了，嵌合蛋白可以被表达、纯化和结晶。噬菌体展示的αRep 选择假设至少应该产生少量的靶蛋白作为选择的诱饵，而在某些情况下，这可能很困难。因此，我们将αRep 文库转移到了一种新的构建体中，该构建体适应于蛋白质互补测定法 (PCA) 的选择。这种新方法允许通过在细菌细胞质中与靶蛋白直接相互作用来选择特异性结合剂，因此不需要预先纯化靶蛋白。通过 PCA 或噬菌体展示选择的αRep 结合剂可用于增强那些难以表达、纯化和/或结晶的蛋白质的表达、稳定性、溶解度和结晶发生。
