Bioluminescence Immunoassay for Cortisol Using Recombinant Aequorin as a Label

2002 ◽  
Vol 306 (2) ◽  
pp. 204-211 ◽  
Author(s):  
Mara Mirasoli ◽  
Sapna K. Deo ◽  
Jennifer C. Lewis ◽  
Aldo Roda ◽  
Sylvia Daunert
Keyword(s):  
Recombinant Aequorin

scholarly journals Requirement of the C-terminal proline residue for stability of the Ca2+-activated photoprotein aequorin

1993 ◽  
Vol 293 (1) ◽  
pp. 181-185 ◽  
Author(s):  
N J Watkins ◽  
A K Campbell
Keyword(s):  
Light Emission ◽  
Specific Activity ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Proline Residue ◽  
Long Term Stability ◽  
Recombinant Aequorin

cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.

scholarly journals Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators

1990 ◽  
Vol 270 (2) ◽  
pp. 309-312 ◽  
Author(s):  
O Shimomura ◽  
S Inouye ◽  
B Musicki ◽  
Y Kishi
Keyword(s):  
Luminescence Intensity ◽  
Intensity Ratio ◽  
Synthetic Analogues ◽  
Chromatographic Behaviour ◽  
Recombinant Aequorin

Properties of a recombinant aequorin were investigated in comparison with those of natural aequorin. In chromatographic behaviour the recombinant aequorin did not match any of ten isoaequorins tested, although it was very similar to aequorin J. Its sensitivity to Ca2+ was found to be higher than that of any isoaequorin except aequorin D. The recombinant aequorin exhibited no toxicity when tested in various kinds of cells, even where samples of natural aequorin had been found to be toxic. Properties of four recombinant semi-synthetic aequorins (fch-, hcp-, e- and n-types), prepared from the recombinant apo-aequorin and synthetic analogues of coelenterazine, were approximately parallel with those of corresponding semi-synthetic aequorins prepared from natural apo-aequorin. Both recombinant e-aequorin and natural e-aequorin J luminesced with high values of the luminescence intensity ratio I400/I465, although the ratios were not pCa-dependent. The recombinant aequorin and recombinant semi-synthetic aequorins are highly suited for monitoring cellular Ca2+.

A solid-phase assay for β-1,4-galactosyltransferase activity in human serum using recombinant aequorin

1991 ◽  
Vol 194 (1) ◽  
pp. 185-191 ◽  
Author(s):  
Paolo F. Zatta ◽  
Kwame Nyame ◽  
Milton J. Cormier ◽  
Sharon A. Mattox ◽  
Pedro A. Prieto ◽  
...  
Keyword(s):  
Human Serum ◽  
Solid Phase ◽  
Recombinant Aequorin ◽  
Solid Phase Assay

Stabilisation of Recombinant Aequorin by Polyols: Activity, Thermostability and Limited Proteolysis

2013 ◽  
Vol 170 (2) ◽  
pp. 273-280 ◽  
Author(s):  
Mehdi Zeinoddini ◽  
Khosro Khajeh ◽  
Saman Hosseinkhani ◽  
Ali Reza Saeedinia ◽  
Seyed-Mortaza Robatjazi
Keyword(s):  
Limited Proteolysis ◽  
Recombinant Aequorin

Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin

Nature ◽  
1992 ◽  
Vol 358 (6384) ◽  
pp. 325-327 ◽  
Author(s):  
Rosario Rizzuto ◽  
Alec W. M. Simpson ◽  
Marisa Brini ◽  
Tullio Pozzan
Keyword(s):  
Rapid Changes ◽  
Recombinant Aequorin
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