Development of a Ca(2+)-activated photoprotein, Photina, and its application to high-throughput screening.
The present work describes the engineering and characterization of a new Ca(2+)-activated photoprotein (Photina) and its use in mammalian cell lines for implementation of flash luminescence cell-based assays for high-throughput screening (HTS). When used to measure the activation of 2 G protein-coupled receptors (GPCRs), targeting Photina to the mitochondria increased the signal strength as compared to the normal cytoplasmic expression of Photina. The mitochondrial-targeted Photina also produced a higher signal-to-noise ratio than conventional calcium dyes and a consistently stronger signal than aequorin when tested under equivalent conditions. MitoPhotina provided strong and reliable results when used to measure the activity of purinergic receptors endogenously expressed in the Chinese Hamster Ovary cells and heterologously expressed GPCRs in response to their cognate ligands. Several different types of flash luminescence plate readers (FLIPR(3), FLIPR(TETRA), CyBi-Lumax flash HT, Lumilux, Lumibox) in different plate formats (96, 384, 1536 wells) were used to validate the use of Photina in HTS. The cell number had to be adjusted to correspond to the qualities of the different readers, but once so adjusted, it provided equivalent results on each device. The results obtained show robust and reproducible light signals that offer new possibilities for application of photoproteins to the generation of cell-based assays for HTS.
Bovolenta
Silvia
S
Axxam SpA, San Raffaele Biomedical Science Park, Milan, Italy.
Foti
Maria
M
Lohmer
Stefan
S
Corazza
Sabrina
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000331:Aequorin / Q000032:analysis / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival; D054428:Chemokine CX3CL1 / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D006632:Histamine / Q000494:pharmacology; D007093:Imidazoles / Q000378:metabolism; D020128:Inhibitory Concentration 50; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011719:Pyrazines / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D011983:Receptors, Purinergic / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D014162:Transfection
D054428:Chemokine CX3CL1; D007093:Imidazoles; D008164:Luminescent Proteins; D011719:Pyrazines; D043562:Receptors, G-Protein-Coupled; D011983:Receptors, Purinergic; D011994:Recombinant Proteins; C017144:coelenterazine; D000331:Aequorin; D006632:Histamine; D000255:Adenosine Triphosphate; D002118:Calcium