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1.13.12.6: Cypridina-luciferin 2-monooxygenase

This is an abbreviated version!
For detailed information about Cypridina-luciferin 2-monooxygenase, go to the full flat file.

Word Map on EC 1.13.12.6

Reaction

Cypridina luciferin
+
O2
=
Oxidized Cypridina luciferin
+
CO2
+
hnu

Synonyms

Apogon luciferase, Apogon luciferase 1, CLase, CLuc, Cypridina luciferase, Cypridina luciferin 2-monooxygenase, Cypridina noctiluca luciferase, Cypridina-type luciferase, Fbp, FBP-IgG, luciferase (Cypridina luciferin), PGE2-luciferase, Vargula luciferase

ECTree

     1 Oxidoreductases
         1.13 Acting on single donors with incorporation of molecular oxygen (oxygenases)
             1.13.12 With incorporation of one atom of oxygen (internal monooxygenases or internal mixed-function oxidases)
                1.13.12.6 Cypridina-luciferin 2-monooxygenase

Purification

Purification on EC 1.13.12.6 - Cypridina-luciferin 2-monooxygenase

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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
biotinylated enzyme is incubated with 1 microM sodium metaperiodate in 0.1 M sodium acetate buffer (pH 5.2), reaction stopped by glycerol addition, loading onto a size exclusion column, oxidized biotinylated enzyme eluted with the same buffer, pooling of bioluminescent fractions, concentration by ultrafree-0.5 centrifugal filter device, addition of 10 mM HiLyte Fluor 647 hydrazine in 0.1 M sodium acetate buffer (pH 5.2), after incubation loading onto a size-exclusion column, elution with 0.1 M potassium phosphate buffer (pH 7.2) containing 0.15 M NaCl. Treatment of human Delta-like protein (Dlk-1) antibody with 2-mercaptoethylamine (cleaving hinge-region disulfide bonds between heavy chains of antibody molecules) and conjugation of half antibodies to maleimide-activated avidin and purification on a size-exclusion column results in a 200 kDa conjugate
-
Escherichia coli cell culture with expression vector is cooled on ice-water bath, expression induced, incubation for 18 h at 15°C, cell concentration by centrifugation, solution in 50 mM Tris-HCl (pH 7.6) and 10 mM EDTA, disruption by sonication, centrifugation, SDS-PAGE
-
loading of recombinant prostaglandin E2 luciferase onto size-exclusion column, elution with 0.1 M potassium phosphate buffer, pH 7.2, and 0.15 M NaCl, fractions with bioluminescent activities (Cypridina luciferin solution, 0.1 M Tris-HCl, pH 7.4, 0.3 M sodium ascorbate, 0.2 M Na2SO3) are combined and stored at -30°C
-
native enzyme by fractional precipitation with acetone and ammonium sulfate, and dialysis, followed by gel filtration
-
native enzyme from photogenic organs by fractional precipitation with acetone and ammonium sulfate, and dialysis, followed by gel filtration
-
Ni affinity column chromatography, SA beads column chromatography, monomeric avidin column chromatography, and TSK G300SW gel filtration
-
recombinant secreted enzyme from Nicotiana benthamiana BY-2 cell medium by ultrafiltration, buffer exchange gel filtration, anion exchange chromatography, and dialysis