Slow luminescence kinetics of semi-synthetic aequorin: expression, purification and structure determination of cf3-aequorin.

cf3-Aequorin is one of the semi-synthetic aequorins that was produced by replacing 2-peroxycoelenterazine (CTZ-OOH) in native aequorin with a 2-peroxycoelenterazine analog, and it was prepared using the C2-modified trifluoromethyl analog of coelenterazine (cf3-CTZ) and the histidine-tagged apoaequorin expressed in Escherichia coli cells. The purified cf3-aequorin showed a slow luminescence pattern with half-decay time of maximum intensities of luminescence of 5.0 s. This is much longer than that of 0.9 s for native aequorin, and its luminescence capacity was estimated to be 72.8% of that of native aequorin. The crystal structure of cf3-aequorin was determined at 2.15 Å resolution. The light source of 2-peroxytrifluoromethylcoelenterazine (cf3-CTZ-OOH) was stabilized by the hydrogen-bonding interactions at the C2-peroxy moiety and the p-hydroxy moiety at the C6-phenyl group. In native aequorin, three water molecules contribute to stabilizing CTZ-OOH through hydrogen bonds. However, cf3-aequorin only contained one water molecule, and the trifluoromethyl moiety at the C2-benzyl group of cf3-CTZ-OOH interacted with the protein by van der Waals interactions. The slow luminescence kinetics of cf3-aequorin could be explained by slow conformational changes due to the bulkiness of the trifluoromethyl group, which might hinder the smooth cleavage of hydrogen bonds at the C2-peroxy moiety after the binding of Ca2+ to cf3-aequorin.

The use of aequorin and its variants for Ca2+ measurements.

Ca(2+)-sensitive photoproteins are ideal agents for measuring the Ca(2+) concentration ([Ca(2+)]) in intracellular organelles because they can be modified to include specific targeting sequences. Aequorin was the first Ca(2+)-sensitive photoprotein probe used to measure the [Ca(2+)] inside specific intracellular organelles in intact cells. Aequorin is a 22-kDa protein produced by the jellyfish Aequorea victoria. On the binding of Ca(2+) to three high-affinity sites in aequorin, an irreversible reaction occurs in which the prosthetic group is released and a photon is emitted. Aequorin has become widely used for intracellular Ca(2+) measurements because it offers many advantages: For example, it can be targeted with precision, functions over a wide range of [Ca(2+)], and shows low buffering capacity. In this article we describe the main characteristics of the aequorin probe and review the reasons why it is widely used to measure intracellular [Ca(2+)].

Using targeted variants of aequorin to measure Ca2+ levels in intracellular organelles.

Aequorin is a Ca(2+)-sensitive photoprotein isolated from the jellyfish Aequorea victoria. It is an ideal probe for measuring Ca(2+) concentration ([Ca(2+)]) in intracellular organelles because it can be modified to include specific targeting sequences. On the binding of Ca(2+) to three high-affinity sites in aequorin, an irreversible reaction occurs in which the prosthetic group coelenterazine is released and a photon is emitted. This protocol presents procedures for expressing, targeting, and reconstituting aequorin in intact and permeabilized mammalian cells and describes how to use this photoprotein to measure intracellular [Ca(2+)] in various subcellular compartments.

Bioluminescence imaging: a shining future for cardiac regeneration.

Advances in bioanalytical techniques have become crucial for both basic research and medical practice. One example, bioluminescence imaging (BLI), is based on the application of natural reactants with light-emitting capabilities (photoproteins and luciferases) isolated from a widespread group of organisms. The main challenges in cardiac regeneration remain unresolved, but a vast number of studies have harnessed BLI with the discovery of aequorin and green fluorescent proteins. First described in the luminous hydromedusan Aequorea victoria in the early 1960s, bioluminescent proteins have greatly contributed to the design and initiation of ongoing cell-based clinical trials on cardiovascular diseases. In conjunction with advances in reporter gene technology, BLI provides valuable information about the location and functional status of regenerative cells implanted into numerous animal models of disease. The purpose of this review was to present the great potential of BLI, among other existing imaging modalities, to refine effectiveness and underlying mechanisms of cardiac cell therapy. We recount the first discovery of natural primary compounds with light-emitting capabilities, and follow their applications to bioanalysis. We also illustrate insights and perspectives on BLI to illuminate current efforts in cardiac regeneration, where the future is bright.

Purification of histidine-tagged aequorin with a reactive cysteine residue for chemical conjugations and its application for bioluminescent sandwich immunoassays.

Highly purified histidine-tagged aequorin with a reactive cysteine residue (His-Cys4-aequorin) was obtained from the periplasmic space of Escherichia coli cells by nickel-chelate affinity chromatography and hydrophobic chromatography. The procedure yielded 40.3mg of His-Cys4-aequorin from 2L of cultured cells with over 95% purity. The chemical conjugates of His-Cys4-aequorin with maleimide-activated streptavidin and maleimide-activated biotin were prepared without significant loss of luminescence activity and were applied to the bioluminescent sandwich immunoassay for α-fetoprotein (AFP) as a model analyte. The measurable range of AFP by these conjugates was 0.01-100 ng/ml and the sensitivities were similar to that using aequorin-labeled specific antibody and amino-biotinylated aequorin.

Early history, discovery, and expression of Aequorea green fluorescent protein, with a note on an unfinished experiment.

The bioluminescent hydromedusan jellyfish, Aequorea victoria, emits a greenish light (lambda(max) = 508 nm) when stimulated electrically or mechanically. The light comes from photocytes located along the margin of its umbrella. The greenish light depends on two intracellular proteins working in consort: aequorin (21.4 kDa) and a green fluorescent protein (27 kDa). An excited state green fluorescent protein molecule results, which, on returning to the ground state, emits a greenish light. Similarly, a green light emission may be induced in the green fluorescent protein by exposing it to ultraviolet or blue light. Because the green light can be readily detected under a fluorescence microscope, the green fluorescent protein, tagged to a protein of interest, has been used widely as a marker to locate proteins in cells and to monitoring gene expression. This article reviews the work that took place leading to the discovery, cloning, and expression of the green fluorescent protein, with a note on an unfinished experiment.

Reconstitution of blue fluorescent protein from recombinant apoaequorin and synthetic coelenteramide.

Blue fluorescent protein of aequorin (BFP) is a complex of Ca(2+)-bound apoaequorin with coelenteramide and is a bifunctional protein, which shows blue fluorescence and the luminescence activity like a luciferase. To reconstitute synthetic BFP (syn-BFP) from apoaequorin and coelenteramide, we established new synthetic route of coelenteramide and prepared highly purified recombinant aequorin using the histidine-tagged secretion system in Escherichia coli cells. As a result, we succeeded in reconstituting syn-BFP quantitatively and the fluorescence and luminescence properties of syn-BFP were identical to that of BFP obtained from aequorin.

Chromatography of isoforms of recombinant apoaequorin and method for the preparation of aequorin.

Gradient elution chromatography of recombinant apoaequorin carried out in the presence of Ca2+ revealed two isoforms of apoaequorin, reduced and oxidized, whereas in the presence of EDTA 3 isoforms were observed. In a regeneration mixture of apoaequorin, coelenterazine, EDTA, and 2-mercaptoethanol, four isoforms were obtained, of which only one, aequorin, gave light with Ca2+. A method is described for the preparation of highly pure aequorin. The aequorin was stable in solution for approximately 10 days at 4 degrees C and pH 7.6, and then it gradually lost activity with a half-life of about 20 days until it was almost completely inactive on day 30.

One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography.

A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form of inclusion bodies. Inclusion bodies were solubilized with urea, followed by purification and refolding of (His)(6)-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose. The purity, as determined by SDS-PAGE analysis, was greater than 80%. The yield was 0.7-1 mg apoaequorin from a 50 ml bacterial culture. The kinetics of light emission of purified aequorin upon addition of Ca(2+) was typical of the commercial aequorin. The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude. As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8.

Bacterial expression of in vivo-biotinylated aequorin for direct application to bioluminometric hybridization assays.

We have constructed a plasmid suitable for bacterial expression of in vivo-biotinylated photoprotein aequorin. The biotin tag facilitates the isolation of aequorin from crude cell extract and the direct complexation of aequorin with streptavidin for the development of highly sensitive hybridization assays, thereby avoiding the need for chemical crosslinking. The plasmid contains a biotin-acceptor coding sequence fused to an apoaequorin gene. The birA gene, encoding biotin protein ligase (BPL), is inserted downstream of the apoaequorin sequence. BPL biotinylates, posttranslationally, the acceptor domain at a unique position. Functional aequorin is generated by incubating the lysate with coelenterazine and is purified by using a monomeric avidin column that allows elution under nondenaturing conditions. The biotinylated aequorin is complexed with streptavidin and used as a reporter molecule in a hybridization assay. The assay entails immobilization of an oligonucleotide probe on microtiter wells followed by hybridization with a denatured DNA target labeled with biotin through PCR. Streptavidin-biotinylated aequorin is used for quantification of the hybrids. Luminescence is measured in the presence of excess Ca(2+). The analytical range extends from 80 amol of target DNA per well (with a signal-to-background ratio of 2.1) up to 40 fmol per well. The coefficient of variation is about 6%. In vivo-biotinylated aequorin produced from 1 liter of culture is sufficient for 300,000 hybridization assays.

The in situ regeneration and extraction of recombinant aequorin from Escherichia coli cells and the purification of extracted aequorin.

Recombinant apoaequorin expressed in the periplasmic space of Escherichia coli cells was regenerated into aequorin and extracted from the cells, simultaneously, using a buffer that contained coelenterazine. Due to the mild extraction conditions, the impurities in the extract were minimal. Thus, the purification of extracted aequorin could be accomplished in only two steps, anion-exchange chromatography and hydrophobic interaction chromatography, simply by adsorption and elution in both steps. The purified recombinant aequorin was pure, based on various data, including HPLC analysis and light-emitting activity. The yield of purified aequorin was 25-35 mg from 600 ml of culture, which was over 75% of the total amount of apoaequorin expressed in E. coli cells.

Construction of a full-length Ca2+-sensitive adenylyl cyclase/aequorin chimera.

Ca2+-sensitive adenylyl cyclases are key integrators of Ca2+ and cAMP signaling. To selectively probe dynamic changes in [Ca2+]i at the plasma membrane where adenylyl cyclases reside, a full-length, Ca2+-inhibitable type VI adenylyl cyclase/aequorin chimera has been constructed by a two-stage polymerase chain reaction method. The expressed adenylyl cyclase/aequorin chimera was appropriately localized to the plasma membrane, as judged by biochemical fractionation and functional analysis. The chimera retained full adenylyl cyclase activity and sensitivity to inhibition by physiological [Ca2+]i elevation. The aequorin portion of the chimeric construct was also capable of measuring changes in [Ca2+] both in vitro and in vivo. When the plasma membrane-tagged aequorin and cytosolic aequorin were compared in their measurement of [Ca2+]i, they showed contrasting sensitivities depending on whether the [Ca2+]i originated from internal stores or capacitative entry. This is the first full-length enzyme-aequorin chimera that retains the full biological properties of both aequorin and a Ca2+-sensitive adenylyl cyclase. This novel chimeric Ca2+ sensor provides the unique ability to directly report the dynamics of [Ca2+]i that regulates this Ca2+-sensitive enzyme under a variety of physiological conditions. Since this chimera is localized to the plasma membrane, it can also be used to assess local changes in [Ca2+]i at the plasma membrane as distinct from global changes in [Ca2+]i within the cytosol.

High-level expression and purification of apoaequorin.

A fairly rapid and improved method for producing large amounts of highly pure apoaequorin, the apoprotein of aequorin which emits light on binding Ca2+, is described. The method consists of fusing the gene of the outer membrane protein A (ompA) secretion signal peptide of Escherichia coli to the apoaequorin gene and expressing the fused gene in the bacterium. The expressed protein is correctly cleaved in the process of being secreted across the cell membrane into the culture medium. The apoaequorin is subsequently purified by acid precipitation and DEAE-cellulose chromatography, yielding a product of greater than 95% purity. The availability of pure apoaequorin makes possible detailed studies of the physical-chemical properties of this Ca2(+)-binding protein and allows for the preparation of pure aequorin for use in highly specific and sensitive assays for Ca2+.

Overexpression and purification of the recombinant Ca2+-binding protein, apoaequorin.

The small, monomeric Ca2+-binding photoprotein, aequorin, emits blue light by an intramolecular reaction when mixed with Ca2+. The photoprotein is made up of coelenterazine and molecular oxygen, bound noncovalently to apoaequorin (apoprotein). The chemical steps leading to light emission, involving the oxidative degradation of coelenterazine, have been studied extensively, but little is known about the active site and how the molecule catalyzes the oxidation of coelenterazine. The three-dimensional structure of the protein has not been determined and therefore answers to these questions have remained unavailable. The present paper describes a procedure for preparing fairly large amounts of apoaequorin and aequorin for X-ray crystallographic studies. It consists of fusing the apoaequorin cDNA to the signal peptide coding sequence of the outer membrane protein A of Escherichia coli, which is under the control of the lipoprotein promoter. When the cDNA was expressed in E. coli, a large excess of the recombinant protein was produced and released into the culture medium. Purification of the protein was accomplished by acid precipitation and DEAE-cellulose chromatography. The procedure yielded 7.4 mg of recombinant apoaequorin with a purity greater than 95% from 200 ml of culture medium. On regeneration with coelenterazine, the recombinant aequorin was fully active with Ca2+.