Photoprotein Photina

In conclusion, the new chimeric photoprotein Photina exhibits a robust flash luminescence signal in different expression systems, rendering this protein a reporter well-suited for HTS and uHTS applications. 

Development of a Ca(2 - - )-activated photoprotein, Photina, and its appli-cation to high-throughput screening. J. Biomol. Screening 2007, 12,... 

In order to create a very sensitive photoprotein, the sequence and structural similarities as well as the unique features of different well-known photoproteins were carefully analyzed. Based on this study a chimeric photoprotein, named Photina , was designed. Indeed, the region between the first two calcium binding sites of the obelin gene was replaced with the corresponding fragment from the photoprotein clytin, producing a novel photoprotein with improved luminescence. 

In order to verify the activity of the chimeric product, in vitro transcription and translation experiments were performed. Active photoprotein formation was monitored by measuring luminescence from the translation mixture in the presence of coelenterazine after the addition of calcium ions. As shown in Fig. 1, Photina produces an intense luminescence signal in response to calcium stimulation (25 mM CaCU) which is generally higher than that observed with natural photoproteins. 

Functional characterization of the new photoprotein was carried out by biochemical analysis of purified recombinant protein. Different quantities of recombinant Photina ranging from 0.09 to 12.5 ng were charged with 10 0M coelenterazine for 4 h at 4 °C. After incubation, 100 pM CaCl2 was applied and the total RLU (relative light units) was recorded for 10 s using a Berthold Luminometer. As shown in Fig. 3, recombinant Photina gives a consistent signal even when used at very low concentrations. 

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