Journal ArticleDOI
Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity.
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TLDR
Improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins, culminating in a >20-fold increase in contrast.Abstract:
Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells, yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dilute proteins. If the affinity of the tetracysteine peptide could be increased, more stringent dithiol washes should increase the contrast between specific and nonspecific staining. Residues surrounding the tetracysteine motif were randomized and fused to GFP, retrovirally transduced into mammalian cells and iteratively sorted by fluorescence-activated cell sorting for high FRET from GFP to ReAsH in the presence of increasing concentrations of dithiol competitors. The selected sequences show higher fluorescence quantum yields and markedly improved dithiol resistance, culminating in a >20-fold increase in contrast. The selected tetracysteine sequences, HRWCCPGCCKTF and FLNCCPGCCMEP, maintain their enhanced properties as fusions to either terminus of GFP or directly to beta-actin. These improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins.read more
Citations
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The fluorescent toolbox for assessing protein location and function
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Engineering and characterization of a superfolder green fluorescent protein.
Jean-Denis Pedelacq,Stéphanie Cabantous,Timothy H. Tran,Thomas C. Terwilliger,Geoffrey S. Waldo +4 more
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HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis
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References
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A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells.
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Irwin Chen,Alice Y. Ting +1 more
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Biotechnological applications of phage and cell display
TL;DR: This review explains the basis of phage and bacterial surface display, the contributions made by these two leading technologies to biotechnological applications, and focuses mainly on three areas wherephage and cell display have had the greatest impact, namely, antibody engineering, enzyme technology and vaccine development.
Book ChapterDOI
Understanding structure-function relationships in the Aequorea victoria green fluorescent protein.
TL;DR: Learning the physiological role of green fluorescent protein (GFP) and its interaction with aequorin could help understand how to create mutants that exhibit efficiency energy transfer and how to effectively control dimerization.