Recombinant Obelin

A cDNA encoding apoobelin was obtained from O. longissima and sequenced (Illarionov et al., 1995). The deduced amino acid sequence of the apoobelin consists of 195 amino acid residues, with a calculated molecular mass of about 22.2 kDa, closely matching the apoproteins of other Ca2+-sensitive photoproteins such as aequorin from the jellyfish Aequorea (Inouye et al., 1985 Prasher et al., 1985) and clytin from the jellyfish Phialidium gregarium (Inouye and Tsuji, 1993). To obtain recombinant apoobelin, the cDNA encoding apoobelin was expressed in E. coli (Illarionov et al., 2000). The recombinant apoobelin produced was purified and converted into obelin by incubation with coelenterazine in the presence of molecular oxygen and 2-mercaptoethanol or dithioerythritol, as in the case of aequorin. 

Concerning the Ca2+-triggered luminescence of obelin, Deng et al. (2001) reported an interesting observation (Fig. 4.2.2) the luminescence of the recombinant obelin from O. longissima is blue (7max 475 nm), whereas a mutant of this obelin, in which the amino acid residue-92 has been changed from tryptophan to phenylalanine, emits 

Deng et al. (2004a,b) prepared the crystals of the spent obelin (W92F mutant from O. longissima) that had been luminesced with Ca2+, and successfully obtained the X-ray structure of apoobelin as an important information in elucidating the mechanism of the luminescence reaction. 

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Fig. 4.2.1 Luminescence spectra of the Ca2+-triggered light emission of recombinant obelins (dotted lines), and the fluorescence emission spectra of their spent solution after luminescence (solid lines). Left obelin derived from O. geniculata right obelin derived from O. longissima. Reproduced from Markova etal., 2002, with permission from the American Chemical Society.

Illarionov, B. A., et al. (2000). Recombinant obelin cloning and expression of cDNA, purification, and characterization as a calcium indicator. Method. Enzymol. 305 223-249. 

Obelin is a Ca2+-activated bioluminescent photoprotein that has been isolated from the marine polyp Obelia longissima. Binding of calcium ions determines a luminescent emission. The protein consists of 195 amino acid residues [264] and is composed of apoobelin, coelenterazine, and oxygen. As aequorin, it contains three EF-hand Ca2+-binding sites and the luminescent reaction may be the result of coelenterazine oxidation by way of an intramolecular reaction that produces coelenteramide, C02, and blue light. As for aequorin, the luminescent reaction of obelin is sensitive to calcium and the protein was used in the past as an intracellular Ca2+ indicator. More recently, the cloning of cDNA for apoobelin led to the use of recombinant obelin as a label in different analytical systems. 

Bondar, V. S., etal. (1995). Cadmium-induced luminescence of recombinant photoprotein obelin. Biochim. Biophys. Acta 1231 29-32. 

Vysotski, E. S., etal. (1999). Preparation and preliminary study of crystals of the recombinant calcium-regulated photoprotein obelin from the biolu-minescent hydroid Obelia longissima. Acta Cryst. D55 1965-1966. 

In addition there is no problem with obelin availability for the present moment we have the hyper-producing strain E. coli of the protein available, and the technology permitting the obtaining of over 50 mg of a highly purified apoobelin per 1 g of raw cell paste. The recombinant apoprotein is effectively activated with a synthetic coelenterazine and does not differ from a native product in biochemical and bioluminescent properties. The protein is stable when stored in a soluble or lyophilized state. 

Using the genetic approach the biotinylated obelin was obtained. A recombinant apoobelin capable of being biotinylated in vivo in E. coli cells with BirA was constructed by fusing in-frame a synthetic DNA-fragment encoding the artificial biotin acceptor peptide to the N-terminus of the obelin cDNA gene. The application of the fusion protein as a label in immunoassay was demonstrated. ... 

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