Journal
NATURE BIOTECHNOLOGY
Volume 23, Issue 10, Pages 1308-1314Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt1136
Keywords
-
Categories
Funding
- NCRR NIH HHS [RR04050] Funding Source: Medline
- NIGMS NIH HHS [GM72033] Funding Source: Medline
- NINDS NIH HHS [NS27177] Funding Source: Medline
Ask authors/readers for more resources
Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells(1,2), yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dilute proteins(2,3). 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available