Directed Improvement of i-Photina Bioluminescence Properties, an Efficient Calcium-Regulated Photoprotein

Photoproteins are excellent reporter systems because they don’t have virtually background signal. Aequorin is the most wellknown photoprotein. Three improved engineered photoproteins photina, iphotina and cphotina, 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 HighThroughput Screening (HTS). These photoproteins have recently been commercialized by several companies. So we selected iphotina having the highest luminescence signal and good stability in comparison with two others. Subsequently, to produce iPhotina 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 iPhotina bioluminescence to shorter wavelengths, photoprotein luminescence activity of F91Y and W95F mutants was increased and decreased, respectively. Moreover, while Ca2+ sensitivity and decay halflife time were increased in both mutants in comparison with iPhotina, 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 iPhotina for HTS and simultaneous assays.