ACS Chem Biol. 2010 Mar 19;5(3):273-7. doi: 10.1021/cb900284c.
Optical biosensors for short peptide motifs, an important class of biomarkers, have been developed based on "affinity clamps", a new class of recombinant affinity reagents. Affinity clamps are engineered by linking a peptide-binding domain and an antibody mimic domain based on the fibronectin type III scaffold, followed by optimization of the interface between the two. This two-domain architecture allows for the design of allosteric coupling of peptide binding to fluorescence energy transfer between two fluorescent proteins attached to the affinity clamp. Coupled with high affinity and specificity of the underlying affinity clamps and rationally designed mutants with different sensitivity, peptide concentrations in crude cell lysate were determined with a low nanomolar detection limit and over 3 orders of magnitude. Because diverse affinity clamps can be engineered, our strategy provides a general platform to generate a repertoire of genetically encoded, label-free sensors for peptide motifs.
基于“亲和夹”（一种新型重组亲和试剂），已经开发出用于短肽基序（一类重要的生物标志物）的光学生物传感器。亲和夹通过连接一个基于纤连蛋白 III 结构域的肽结合结构域和一个抗体模拟结构域，并对两者之间的界面进行优化而构建。这种双结构域结构允许设计肽结合的变构偶联和附着在亲和夹上的两个荧光蛋白之间的荧光能量转移。再加上基础亲和夹的高亲和力和特异性以及具有不同灵敏度的合理设计突变体，使得可以在粗细胞裂解物中以低纳摩尔检测限和超过 3 个数量级的范围来确定肽浓度。由于可以设计出各种亲和夹，因此我们的策略为生成用于肽基序的基因编码、无标记传感器提供了一个通用平台。
