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.