Cloning and expression of the bioluminescent photoprotein pholasin from the bivalve mollusc Pholas dactylus.
Pholasin is the photoprotein responsible for luminescence in the bivalve Pholas dactylus and consists of a luciferin tightly bound to a glycosylated protein. It is a sensitive indicator of reactive oxygen species. A full-length clone encoding apopholasin was isolated from a P. dactylus light organ cDNA library. The unprocessed apoprotein contained 225 amino acids, starting with a signal peptide of 20 amino acids, 3 predicted N-linked glycosylation sites, 1 O-linked site, no histidines, and 7 cysteines. The recombinant apoprotein was expressed in cell extracts and insect cells. The size of the apoprotein expressed in cell extracts and the cytosol of insect cells was 26 kDa but that of the fully processed protein was 34 kDa, as was native pholasin. Both the processed and unprocessed recombinant apoproteins were recognized by a polyclonal antibody raised against native pholasin. Acid methanol extracts from Pholas added to recombinant apoprotein resulted in chemiluminescence triggered by sodium hypochlorite but not photoprotein formation. These results have important implications in understanding the molecular evolution of bioluminescence and will allow the development of recombinant pholasin as an intracellular indicator of reactive oxygen species.
Dunstan
S L
SL
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom.
Sala-Newby
G B
GB
Fajardo
A B
AB
Taylor
K M
KM
Campbell
A K
AK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D049454:Firefly Luciferin / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D008974:Mollusca; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011994:Recombinant Proteins; D049454:Firefly Luciferin; C052157:pholasin