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Selecting for Fast Protein–Protein Association As Demonstrated on a Random TEM1 Yeast Library Binding BLIP
Biochemistry ( IF 2.9 ) Pub Date : 2018-04-19 00:00:00 , DOI: 10.1021/acs.biochem.8b00172
Ruth Cohen-Khait 1 , Gideon Schreiber 1
Affiliation  

Protein–protein interactions mediate the vast majority of cellular processes. Though protein interactions obey basic chemical principles also within the cell, the in vivo physiological environment may not allow for equilibrium to be reached. Thus, in vitro measured thermodynamic affinity may not provide a complete picture of protein interactions in the biological context. Binding kinetics composed of the association and dissociation rate constants are relevant and important in the cell. Therefore, changes in protein–protein interaction kinetics have a significant impact on the in vivo activity of the proteins. The common protocol for the selection of tighter binders from a mutant library selects for protein complexes with slower dissociation rate constants. Here we describe a method to specifically select for variants with faster association rate constants by using pre-equilibrium selection, starting from a large random library. Toward this end, we refine the selection conditions of a TEM1-β-lactamase library against its natural nanomolar affinity binder β-lactamase inhibitor protein (BLIP). The optimal selection conditions depend on the ligand concentration and on the incubation time. In addition, we show that a second sort of the library helps to separate signal from noise, resulting in a higher percent of faster binders in the selected library. Fast associating protein variants are of particular interest for drug development and other biotechnological applications.

中文翻译:

如随机TEM1酵母库结合BLIP所示,选择快速的蛋白质-蛋白质结合

蛋白质之间的相互作用介导了绝大多数细胞过程。尽管蛋白质相互作用也遵循细胞内的基本化学原理,但体内生理环境可能无法达到平衡。因此,体外测量的热力学亲和力可能无法提供生物学背景下蛋白质相互作用的完整描述。由缔合和解离速率常数组成的结合动力学在细胞中是相关且重要的。因此,蛋白质-蛋白质相互作用动力学的变化对体内有重大影响蛋白质的活性。从突变体文库中选择更紧密结合剂的通用方案是选择具有较慢解离速率常数的蛋白质复合物。在这里,我们描述了一种从大型随机库开始,通过使用预平衡选择来专门选择具有更快关联速率常数的变体的方法。为此,我们针对其天然的纳摩尔亲和力结合剂β-内酰胺酶抑制剂蛋白(BLIP)完善了TEM1-β-内酰胺酶文库的选择条件。最佳选择条件取决于配体浓度和孵育时间。此外,我们显示出第二种库有助于将信号与噪声分离,从而在选定库中产生更高百分比的更快的粘合剂。
更新日期:2018-04-19
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