Ca2+-regulated Photoprotein Obelin as N-terminal Partner in the Fusion Proteins

Full text (.pdf)
Issue
Биология. Biology. 2010 3 (4)
Authors
Eremeeva, Elena V.; Frank, Ludmila A.; Markova, Svetlana V.; Vysotski, Eugene
Contact information
Eremeeva, Elena V. : Institute of Biophysics SB RAS , Russia, 660036, Krasnoyarsk, Akademgorodok; Frank, Ludmila A. : Institute of Biophysics SB RAS Siberian Federal University , Russia, 660036, Krasnoyarsk, Akademgorodok Russia, 660041, Krasnoyarsk, Svobodny av., 79 , e-mail: ; Markova, Svetlana V. : Institute of Biophysics SB RAS Siberian Federal University , Russia, 660036, Krasnoyarsk, Akademgorodok Russia, 660041, Krasnoyarsk, Svobodny av., 79; Vysotski, Eugene : Institute of Biophysics SB RAS , Russia, 660036, Krasnoyarsk, Akademgorodok
Keywords
cgreGFP; obelin; C-end fusing; cgreGFP
Abstract

Ca2+-regulated photoproteins genetically fused with biospecific polipeptides have been extensively used in intracellular Ca2+ 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 Ca2+-regulated photoprotein obelin may be obtained by fusing with biospecific polypeptides regardless its termini.

Pages
372-383
Paper at repository of SibFU
http://elib.sfu-kras.ru/handle/2311/2248

Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).