Designed Proteins as Novel Imaging Reagents in Living Escherichia coli.
Fluorescence imaging is a powerful tool to study protein function in living cells. Here, we introduce a novel imaging strategy that is fully genetically encodable, does not require the use of exogenous substrates, and adds a minimally disruptive tag to the protein of interest (POI). Our method was based on a set of designed tetratricopeptide repeat affinity proteins (TRAPs) that specifically and reversibly interact with a short, extended peptide tag. We co-expressed the TRAPs fused to fluorescent proteins (FPs) and the peptide tags fused to the POIs. We illustrated the method using the Escherichia coli protein FtsZ and showed that our system could track distinct FtsZ structures under both low and high expression conditions in live cells. We anticipate that our imaging strategy will be a useful tool for imaging the subcellular localization of many proteins, especially those recalcitrant to imaging by direct tagging with FPs.
Department of Physics, Yale University, 217 Prospect Street, New Haven, CT, 06511, USA.|Integrated Graduate Program in Physical and Engineering Biology, Yale University, 266 Whitney Avenue, New Haven, CT, 06511, USA.
Susan E
SE
Pratt
Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT, 06511, USA.
Elizabeth B
EB
Speltz
Department of Physics, Yale University, 217 Prospect Street, New Haven, CT, 06511, USA. simon.mochrie@yale.edu.|Integrated Graduate Program in Physical and Engineering Biology, Yale University, 266 Whitney Avenue, New Haven, CT, 06511, USA. simon.mochrie@yale.edu.|Department of Applied Physics, Yale University, 15 Prospect Street, New Haven, CT, 06511, USA. simon.mochrie@yale.edu.
Simon G J
SG
Mochrie
Integrated Graduate Program in Physical and Engineering Biology, Yale University, 266 Whitney Avenue, New Haven, CT, 06511, USA. lynne.regan@yale.edu.|Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT, 06511, USA. lynne.regan@yale.edu.|Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT, 06511, USA. lynne.regan@yale.edu.
Lynne
L
Regan
D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000032:analysis* / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology* / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050296:Microbial Viability; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; C071525:FtsZ protein, Bacteria; D008164:Luminescent Proteins; D010455:Peptides
fluorescence imaging; fluorescent probes; protein design; repeat protein; tag-probe system