Linked Life Data

16155565

Source:http://linkedlifedata.com/resource/pubmed/id/16155565

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
10
pubmed:dateCreated
2005-10-7
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
1087-0156
pubmed:author
pubmed:issnType
Print
pubmed:volume
23
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1308-14
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity.
pubmed:affiliation
Department of Pharmacology, University of California, San Diego, 9500 Gilman Dr., La Jolla, California, 92093-0647, USA.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't, Evaluation Studies, Research Support, N.I.H., Extramural