Rapid development of genetically encoded FRET reporters

Alen Piljić, Iñaki De Diego, Matthias Wilmanns, Carsten Schultz

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

To meet the demand on genetically encoded reporter molecules for live cell imaging, we introduce a new facile combined cloning and FRET reporter analysis strategy. The versatile and fully orthogonal cloning approach involves a set of up to 36 vectors featuring a variety of fluorescent protein FRET pairs and different length linkers. The construct set was successfully applied to two calmodulin-binding proteins, the death-associated protein kinase 1 (DAPK1) and calcium/calmodulin-dependent protein kinase II α (Camk2a). Clone analysis and reporter validation was performed by printing plasmid DNA arrays and subsequent semiautomated microscopy of reversely transfected cells. Characterization of the best performing DAPK1 and Camk2a reporters revealed significant differences in translating calcium signals into kinase responses despite the close functional and structural similarity.

Original languageEnglish (US)
Pages (from-to)685-691
Number of pages7
JournalACS Chemical Biology
Volume6
Issue number7
DOIs
StatePublished - Jul 15 2011
Externally publishedYes

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Death-Associated Protein Kinases
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Cloning
Organism Cloning
Calcium-Calmodulin-Dependent Protein Kinase Kinase
Calmodulin-Binding Proteins
Calcium-Calmodulin-Dependent Protein Kinases
Printing
Oligonucleotide Array Sequence Analysis
Microscopy
Microscopic examination
Plasmids
Phosphotransferases
Clone Cells
Calcium
Imaging techniques
Molecules
DNA
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine

Cite this

Rapid development of genetically encoded FRET reporters. / Piljić, Alen; De Diego, Iñaki; Wilmanns, Matthias; Schultz, Carsten.

In: ACS Chemical Biology, Vol. 6, No. 7, 15.07.2011, p. 685-691.

Research output: Contribution to journalArticle

Piljić, Alen ; De Diego, Iñaki ; Wilmanns, Matthias ; Schultz, Carsten. / Rapid development of genetically encoded FRET reporters. In: ACS Chemical Biology. 2011 ; Vol. 6, No. 7. pp. 685-691.
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