利用体外区室化技术对链霉亲和素变体进行定向进化。
Directed evolution of streptavidin variants using in vitro compartmentalization.
作者信息
Levy Matthew, Ellington Andrew D
机构信息
Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
出版信息
Chem Biol. 2008 Sep 22;15(9):979-89. doi: 10.1016/j.chembiol.2008.07.017.
Abstract
We have developed and implemented an in vitro compartmentalization (IVC) selection scheme for the identification of streptavidin (SA) variants with altered specificities for the biotin analog desthiobiotin. Wild-type SA and selected variants bind desthiobiotin with similar affinities (approximately 10(-13) M), but the variants have off rates almost 50 times slower and a half-life for dissociation of 24 hr at 25 degrees C. The utility of streptavidin variants with altered specificities and kinetic properties was shown by constructing protein microarrays that could be used to differentially organize and immobilize DNAs bearing these ligands. The methods we have developed should prove to be generally useful for generating a variety of novel SA reagents and for evolving other extremely high-affinity protein:ligand couples.
摘要
我们开发并实施了一种体外区室化（IVC）筛选方案，用于鉴定对生物素类似物脱硫生物素具有改变特异性的链霉亲和素（SA）变体。野生型SA和选定的变体以相似的亲和力（约10^(-13) M）结合脱硫生物素，但这些变体的解离速率几乎慢50倍，在25℃下的解离半衰期为24小时。通过构建可用于差异组织和固定携带这些配体的DNA的蛋白质微阵列，展示了具有改变的特异性和动力学性质的链霉亲和素变体的实用性。我们开发的方法应证明对生成各种新型SA试剂以及进化其他极高亲和力的蛋白质：配体对普遍有用。
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