@article{828601aca71b42c6b4f52e25c7978d28,
title = "Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface",
abstract = "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. Limsakul et al. demonstrate that directed evolution and sequence-function analysis are promising tools for engineering molecular binders and hybrid FRET biosensors, which reveal new distinct subcellular features of MT1-MMP molecular regulations at the extracellular surface of live cells.",
keywords = "FRET biosensor, MT1-MMP, R-phycoerythrin, cell-cell contacts, directed evolution, monobody",
author = "Praopim Limsakul and Qin Peng and Yiqian Wu and Allen, {Molly E.} and Jing Liang and Remacle, {Albert G.} and Tyler Lopez and Xin Ge and Kay, {Brian K.} and Huimin Zhao and Strongin, {Alex Y.} and Yang, {Xiang Lei} and Shaoying Lu and Yingxiao Wang",
note = "Funding Information: This work is supported by grants from NIH HL098472, HL109142, HL121365 (Y. Wang), NSF CBET1360341, DMS1361421 (Y. Wang and S.L.), UC San Diego, and Beckman Laser Institute, Inc. (Y. Wang) and NIH NS 085092 (X.L.Y.). This research was also supported by NSF China NSFC 11428207 (Y. Wang). P.L. acknowledges fellowship support from the Thai Ministry of Science and Technology. Q.P. acknowledges fellowship support from China Scholarship Council (CSC). J.L. acknowledges fellowship support from the Agency for Science, Technology and Research of Singapore. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding Information: This work is supported by grants from NIH HL098472 , HL109142 , HL121365 (Y. Wang), NSF CBET1360341 , DMS1361421 (Y. Wang and S.L.), UC San Diego , and Beckman Laser Institute, Inc . (Y. Wang) and NIH NS 085092 (X.L.Y.). This research was also supported by NSF China NSFC 11428207 (Y. Wang). P.L. acknowledges fellowship support from the Thai Ministry of Science and Technology . Q.P. acknowledges fellowship support from China Scholarship Council (CSC). J.L. acknowledges fellowship support from the Agency for Science, Technology and Research of Singapore . The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ",
year = "2018",
month = apr,
day = "19",
doi = "10.1016/j.chembiol.2018.01.002",
language = "English (US)",
volume = "25",
pages = "370--379.e4",
journal = "Cell Chemical Biology",
issn = "2451-9448",
publisher = "Elsevier Inc.",
number = "4",
}