A novel yellow fluorescent protein of recombinant apoPholasin with dehydrocoelenterazine.

Pholasin is classified as a photoprotein and comprises apoPholasin (an apoprotein of pholasin) and an unknown prosthetic group as the light-emitting source. The luminescence reaction of pholasin is triggered by reactive oxygen species. Recombinant apoPholasin was recently expressed as a fusion protein of glutathione S-transferase (GST-apoPholasin) and purified from E. coli cells. By incubating non-fluorescent dehydrocoelenterazine (dCTZ, dehydrogenated form of CTZ) with GST-apoPholasin, the complex of GST-apoPholasin and dCTZ (GST-apoPholasin/dCTZ complex) was formed immediately and showed bright yellow fluorescence (λmax = 539 nm, excited at 430 nm). Unexpectedly, the fluorescent chromophore of the GST-apoPholasin/dCTZ complex was identified as non-fluorescent dCTZ. The luminescence intensity of the GST-apoPholasin/dCTZ complex was increased in a catalase-H2O2 system, but not in sodium hypochlorite.

Streptavidin-aequorin fusion protein for bioluminescent immunoassay.

The fusion protein of streptavidin to aequorin (STA-AQ) was highly purified from inclusion bodies in Escherichia coli cells and applied to a bioluminescent sandwich immunoassay. α-Fetoprotein (AFP), which is a serological marker of liver cancer, was used as a model analyte to test STA-AQ in an immunoassay. The measurable range of AFP by the sandwich immunoassay, using the complex of STA-AQ and the biotinylated anti-AFP antibody, was 0.02-200 ng/mL with an average coefficient of variation of 4.9%. The detection sensitivity with the complex of STA-AQ and the biotinylated anti-AFP antibody was similar to that with the complex of biotinylated aequorin, streptavidin and the biotinylated anti-AFP antibody. STA-AQ would be a useful reporter protein for immunoassays.

Application of new semisynthetic aequorins with long half-decay time of luminescence to G-protein-coupled receptor assay.

Aequorin is a Ca(2+)-binding photoprotein and consists of an apoprotein (apoaequorin) and a 2-peroxide of coelenterazine. Eight new coelenterazine analogues modified at the C2-position were synthesized and incorporated into recombinant apoaequorin with O(2) to yield different semisynthetic aequorins. The luminescence properties and the sensitivity to Ca(2+) of these semisynthetic aequorins were characterized. Two semisynthetic aequorins, namely me- and cf3-aequorin, showed a slow decay of the luminescence pattern with less sensitivity to Ca(2+) and were useful for the cell-based G-protein-coupled receptor (GPCR) reporter assays.

Expression and purification of the calcium binding photoprotein mitrocomin using ZZ-domain as a soluble partner in E. coli cells.

We constructed a vector for soluble protein expression in the cytoplasm of Escherichia coli cells using the cold induced system. The vector, named pCold-ZZ-P-X, consists of a histidine tag sequence, IgG binding domain of protein A (ZZ domain), the cleavage site of human rhinovirus 3C protease followed by the multiple cloning sites under the controlled of the cold shock protein A (cspA) promoter and the lac operator. Using this expression vector, the calcium binding photoprotein mitrocomin from luminous jellyfish was successfully expressed as a soluble ZZ fusion protein and purified. After removing the ZZ domain by protease digestion, recombinant apomitrocomin was obtained and then regenerated to mitrocomin by incubation with coelenterazine. The luminescence properties of recombinant mitrocomin were characterized and compared to other photoproteins including aequorin, clytin-I and clytin-II.

Soluble protein expression in E. coli cells using IgG-binding domain of protein A as a solubilizing partner in the cold induced system.

We constructed a cold induced expression vector in Escherichia coli cells that consists of a histidine tag sequence for nickel chelate affinity purification, IgG-binding domain of protein A (ZZ-domain) and the multiple cloning sites. The role of ZZ-domain as a solubilizing partner at 15 degrees C was demonstrated by expressing the imidazopyrazinone-type luciferases of Renilla, Oplophorus, Gaussia, and Vargula (Cypridina) as well as the calcium-binding photoproteins and firefly luciferase. The fused protein with ZZ-domain was expressed efficiently as a soluble form in the cytoplasm of E. coli cells at low temperature.

Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps.

Gaussia luciferase secreted by the copepod Gaussia princeps catalyzes the oxidation of coelenterazine to produce blue light. The primary structure of Gaussia luciferase deduced from the cDNA sequence shows two repeat sequences of 71 amino acid residues, suggesting the luciferase consists of two structural domains. Two domains in Gaussia luciferase were expressed independently in Escherichia coli cells, purified and characterized. We found that both domains have luminescence activity with coelenterazine, and the catalytic properties including luminescence spectrum, optimal pH, substrate specificity and luminescence stimulation by halogen ions (Cl-, Br- and I-) are identical to intact Gaussia luciferase. Thus, Gaussia luciferase has two catalytic domains for the luminescence reaction.

Expression, purification and characterization of a photoprotein, clytin, from Clytia gregarium.

A novel histidine-tagged secretion vector in Escherichia coli was constructed and large amounts of highly pure clytin, a calcium-binding photoprotein, was prepared. The histidine-tagged apoclytin expressed into the periplasmic space in E. coli was purified by nickel chelate affinity chromatography. Recombinant clytin was regenerated from apoclytin by incubation with coelenterazine in the presence of dithiothreitol and then purified by anion-exchange chromatography and hydrophobic chromatography. The yield of recombinant clytin was 20mg from 2L of cultured cells with purity greater than 95%. Luminescence properties of recombinant clytin were identical to that of native clytin (phialidin). The Ca(2+) sensitivity of recombinant clytin is lower than that of aequorin and clytin is suited for measuring higher concentration of Ca(2+). Semi-synthetic clytins were also prepared with coelenterazine analogues, and the initial intensity, luminescence capacity and half decay time were characterized.