定向进化工程单域抗体用于活细胞表面的 FRET 生物传感器组装和成像。
Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface.
机构信息
Department of Bioengineering, Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
出版信息
Cell Chem Biol. 2018 Apr 19;25(4):370-379.e4. doi: 10.1016/j.chembiol.2018.01.002. Epub 2018 Jan 27.
Monitoring enzymatic activities at the cell surface is challenging due to the poor efficiency of transport and membrane integration of fluorescence resonance energy transfer (FRET)-based biosensors. Therefore, we developed a hybrid biosensor with separate donor and acceptor that assemble in situ. The directed evolution and sequence-function analysis technologies were integrated to engineer a monobody variant (PEbody) that binds to R-phycoerythrin (R-PE) dye. PEbody was used for visualizing the dynamic formation/separation of intercellular junctions. We further fused PEbody with the enhanced CFP and an enzyme-specific peptide at the extracellular surface to create a hybrid FRET biosensor upon R-PE capture for monitoring membrane-type-1 matrix metalloproteinase (MT1-MMP) activities. This biosensor revealed asymmetric distribution of MT1-MMP activities, which were high and low at loose and stable cell-cell contacts, respectively. Therefore, directed evolution and rational design are promising tools to engineer molecular binders and hybrid FRET biosensors for monitoring molecular regulations at the surface of living cells.
由于荧光共振能量转移（FRET）基于生物传感器的运输和膜整合效率低下，因此在细胞表面监测酶活性具有挑战性。因此，我们开发了一种具有单独供体和受体的混合生物传感器，它们可原位组装。定向进化和序列功能分析技术被整合在一起，以设计一种与藻红蛋白（R-PE）染料结合的单域抗体变体（PEbody）。PEbody 用于可视化细胞间连接的动态形成/分离。我们进一步将 PEbody 与增强型 CFP 和细胞外表面的酶特异性肽融合，在 R-PE 捕获时创建混合 FRET 生物传感器，以监测膜型 1 基质金属蛋白酶（MT1-MMP）的活性。该生物传感器揭示了 MT1-MMP 活性的不对称分布，在松散和稳定的细胞-细胞接触处分别为高和低。因此，定向进化和合理设计是工程分子结合物和混合 FRET 生物传感器以监测活细胞表面分子调节的有前途的工具。
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