%0 Journal Article %T Directed Improvement of i-Photina Bioluminescence Properties, an Efficient Calcium-Regulated Photoprotein %J Biomacromolecular Journal %I Iran Society of Biophysical Chemistry (ISOBC) %Z 7280-2423 %A Ahmadi, Fatemeh %A Sajedi, Reza %A Mahdavi, Atiyeh %A Zeinoddini, Mehdi %A Taghdir, Majid %D 2015 %\ 07/01/2015 %V 1 %N 1 %P 80-92 %! Directed Improvement of i-Photina Bioluminescence Properties, an Efficient Calcium-Regulated Photoprotein %K Photoprotein %K i-Photina %K Bioluminescence emission spectrum %K HTS (High-Throughput Screening) %R %X   Photoproteins are excellent reporter systems because they don’t have virtually background signal. Aequorin is the most well-known photoprotein. Three improved engineered photoproteins photina, i-photina and c-photina, were also recently developed and optimized for generation of Ca2+ mobilization assays precisely. The total light emission is greater than aequorin and their reaction kinetics is also lower. Thus they have improved the applications of flash luminescence assays in High-Throughput Screening (HTS). These photoproteins have recently been commercialized by several companies. So we selected i-photina having the highest luminescence signal and good stability in comparison with two others. Subsequently, to produce i-Photina variants with improved analytical properties such as alternative emission colors, two mutants (F91Y and W95F mutants) were prepared by using site directed mutagenesis. Results showed as both substitutions shifted i-Photina bioluminescence to shorter wavelengths, photoprotein luminescence activity of F91Y and W95F mutants was increased and decreased, respectively. Moreover, while Ca2+ sensitivity and decay half-life time were increased in both mutants in comparison with i-Photina, F91Y mutant presented more stability and higher bioluminescence activity. So, F91Y mutant is an improved version of photoproteins that in many ways is superior to the other Ca2+ indicators such as aequorin and i-Photina for HTS and simultaneous assays.   %U https://www.bmmj.org/article_12815_cc11c97140271ef5a4c179b77ab79279.pdf