Single fluorescent protein-based Ca2+sensors with increased dynamic range

Springer Science and Business Media LLC - Tập 7 Số 1 - 2007
Ekaterina A. Souslova1, Vsevolod V. Belousov1, John G. Lock2, Staffan Strömblad2, Sergey Kasparov3, A D Bolshakov4, V. G. Pinelis4, Yulii A. Labas5, Sergey Lukyanov1, Lorenz M. Mayr6, Dmitriy M. Chudakov1
1Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Miklukho-Maklaya 16/10, 117997, Moscow, Russia
2Karolinska Institutet, Department of Biosciences and Nutrition, Novum, SE-141 57, Huddinge, Sweden
3Department of Physiology School of Medical Sciences, University of Bristol, BS8 1TD, Bristol, UK
4Scientific Centre for Children's Health RAMS, Lomonosovskii pr. 61/2, Moscow, Russia
5Bakh Institute of Biochemistry, RAS, Leninsky 33, 117071, Moscow, Russia
6Novartis Pharma AG, NIBR/DT/LDC, Lichtstrasse 35, CH-4002, Basel, Switzerland

Tóm tắt

Abstract Background Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP). Results Here we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons. Conclusion We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.

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