Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process.
Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process.
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DOI：
10.1093/protein/gzv029
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发表时间：
2015-09
期刊：
影响因子：
--
通讯作者：
Hollfelder F
中科院分区：
文献类型：
--
作者：
Houlihan G;Gatti-Lafranconi P;Lowe D;Hollfelder F
体外展示技术已被证明是获得高亲和力蛋白质结合体的有力工具。我们最近描述了SNAP Display，这是一个完全在体外的DNA展示系统，它使用SNAP标签在油包水乳剂中将蛋白质与其编码的DNA连接起来。在这里，我们应用SNAP显示器来亲和成熟设计的锚蛋白重复蛋白(DARPin)，它与先前通过核糖体展示分离的HER2的胞外结构域结合。经过四次SNAP展示选择循环后，分离出了在体外与HER2特异结合的蛋白质，其解离常数在低到亚纳摩尔范围内。在体外，进化的DARPins组的亲和力与乳腺癌细胞系上与HER2结合的进化的DARPins的荧光强度直接相关。与作为亲和力成熟起点的亲本DARPin相比，最改进的DARPin的热稳定性降低了，因此观察到了稳定性的权衡。对亲和力最高的变异体DARPin F1的框架突变的剖析表明，在四轮进化过程中，功能不稳定和补偿性突变累积。
In vitro display technologies have proved to be powerful tools for obtaining high-affinity protein binders. We recently described SNAP display, an entirely in vitro DNA display system that uses the SNAP-tag to link protein with its encoding DNA in water-in-oil emulsions. Here, we apply SNAP display for the affinity maturation of a designed ankyrin repeat proteins (DARPin) that binds to the extracellular domain of HER2 previously isolated by ribosome display. After four SNAP display selection cycles, proteins that bound specifically to HER2 in vitro, with dissociation constants in the low- to sub-nanomolar range, were isolated. In vitro affinities of the panel of evolved DARPins directly correlated with the fluorescence intensities of evolved DARPins bound to HER2 on a breast cancer cell line. A stability trade-off is observed as the most improved DARPins have decreased thermostability, when compared with the parent DARPin used as a starting point for affinity maturation. Dissection of the framework mutations of the highest affinity variant, DARPin F1, shows that functionally destabilising and compensatory mutations accumulated throughout the four rounds of evolution.
雌激素受体分析在乳腺癌早期癌中的长期预后意义。
影响因子：
8.8
作者：
Winstanley, J;Cooke, T;George, W D;Murray, G;Holt, S;Croton, R;Griffiths, K;Nicholson, R
通讯作者：
Nicholson, R