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 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.