Ca<sup>2+</sup>-regulated Photoprotein Obelin as N-terminal Partner in the Fusion Proteins
2010
Elena V. Eremeeva | Ludmila A. Frank | Svetlana V. Markova | Eugene Vysotski
Ca<sup>2+</sup>-regulated photoproteins genetically fused with biospecific polipeptides have been extensively used in intracellular Ca<sup>2+</sup> measurements and in the development of binding assays. Gene fusions to aequorin have been limited to its N-terminus, since as previous studies indicated the protein loses bioluminescent activity upon modification of its C-terminus. To investigate this in regard to another photoprotein - obelin (OL) the one was elongated at its C-terminus with tyrosine (Y) and then fused with green fluorescent protein Clytia gregaria (cgreGFP) through the flexible 31 aa linker. Both proteins (OL-Y and OL-cgreGFP) were isolated and investigated. The OL-Y was found to form a stable photoprotein comlex, possessing 75 % of WT-OL bioluminescent activity. OL-cgreGFP activity preserves 46 % of WT-OL activity and demonstrates an effective resonance energy transfer, where OL-partner and cgreGFP-partner are energy donor and acceptor respectively. Thus, it was shown that the labels on the base of Ca<sup>2+</sup>-regulated photoprotein obelin may be obtained by fusing with biospecific polypeptides regardless its termini.
显示更多 [+] 显示较少 [-]