Biomedical subjects

R O McCann

Publications and source records attributed to R O McCann.

6 recordsLinked to original sources

PubMed1992

Use of recombinant biotinylated aequorin in microtiter and membrane-based assays: purification of recombinant apoaequorin from Escherichia coli.

Aequorin is a calcium-dependent bioluminescent protein isolated from the hydromedusan Aequorea victoria. The gene for aequorin has been cloned and overexpressed in Escherichia coli [Prasher et al. (1985) Biochem. Biophys. Res. Commun. 126, 1259; Prasher et al. (1987) Biochemistry 26, 1326]. Higher levels of expression have recently been obtained by subcloning aequorin cDNA into the pRC23 plasmid vector such that its expression is under control of the lambda PL promoter [Cormier et al. (1989) Photochem. Photobiol. 49, 509]. Purification of recombinant apoaequorin from E. coli containing this new recombinant plasmid (pAEQ1.3) was accomplished by a two-step procedure involving gel filtration and anion-exchange chromatography on Sephadex G-100 and DEAE-Sepharose, respectively. Typically, 400-500 mg of recombinant protein was obtained from 100 L of fermentation culture. The purified recombinant apoaequorin could be converted to aequorin in high yield upon incubation with synthetic coelenterate luciferin, dissolved oxygen, and a thiol reagent with a photon yield similar to the native photoprotein. Detection of recombinant aequorin in the Dynatech ML1000 Microplate luminometer was linear between 10(-18) and 10(-12) mol, and little loss of specific activity was observed when the protein was derivatized with biotin. The biotinylated derivative was stable when frozen, lyophilized, or stored at 4 degrees C. The feasibility of using biotinylated aequorin as a nonradioactive tag was established by its application in a variety of solid-phase assay formats using the high-affinity streptavidin/biotin interaction. A microtiter-based bioluminescent immunoassay (BLIA) using biotinylated aequorin and the ML1000 luminometer was developed for the detection of subnanogram amounts of a glycosphingolipid (Forsmann antigen). In addition, nanogram to subnanogram quantities of protein antigens and DNA, immobilized on Western and Southern blots, respectively, were detected on instant and X-ray films using biotinylated aequorin.

Aequorin↗

PubMed1991

Isolation and expression of a cDNA encoding Renilla reniformis luciferase.

Renilla reniformis is an anthozoan coelenterate capable of exhibiting bioluminescence. Bioluminescence in Renilla results from the oxidation of coelenterate luciferin (coelenterazine) by luciferase [Renilla-luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5]. In vivo, the excited state luciferin-luciferase complex undergoes the process of nonradiative energy transfer to an accessory protein, green fluorescent protein, which results in green bioluminescence. In vitro, Renilla luciferase emits blue light in the absence of any green fluorescent protein. A Renilla cDNA library has been constructed in lambda gt11 and screened by plaque hybridization with two oligonucleotide probes. We report here the isolation and characterization of a luciferase cDNA and its gene product. The recombinant luciferase expressed in Escherichia coli is identical to native luciferase as determined by SDS/PAGE, immunoblot analysis, and bioluminescence emission characteristics.

Amino Acid Sequence↗

PubMed1989

The enzymology and molecular biology of the Ca2+-activated photoprotein, aequorin.

Aequorin is a bioluminescent protein, isolated from the hydromedusan Aequorea victoria. A recombinant cDNA plasmid (pAEQ1) was shown to encode apoaequorin by detecting photoprotein activity in an extract of an E. coli strain containing pAEQ1 (Prasher et al., 1986, Biochem. Biophys. Res. Comm. 126, 1259-1268). The nucleotide sequence of the pAEQ1 insert has been determined and is shown to differ significantly from the aequorin cDNA (AQ440) isolated by Inouye et al. (1985, Proc. Natl. Acad. Sci. USA 82, 3154-3158). Comparisons of the coding regions of the two cDNAs show there are 52 nucleotide differences, 19 of which are responsible for 18 amino acid replacements. These differences explain the microheterogeneity observed at 17 positions during the sequencing of native apoaequorin. Five aequorin isotypes extracted from Aequorea tissue are observed on 2-dimensional gels and the E. coli-expressed apoaequorin is shown to co-migrate with one of these isotypes. The multiple isotypes could be caused by the presence of a multi-gene family since Southern blot analysis of Aequorea DNA suggests the presence of a minimum of four aequorin genes. Immunoblot analysis suggests that purified native aequorin is proteolytically cleaved during its purification from Aequorea. Comparison of the deduced cDNA translations and the protein sequence suggests the loss of seven residues from the amino terminal. Overexpression of the apoaequorin cDNA in E. coli now provides the means of obtaining gram quantities of a single isotype of the protein which can be converted to aequorin in the presence of coelenterate luciferin, oxygen and an appropriate thiol. Proper extraction procedures and a single chromatographic step provides apoaequorin which is greater than 95% homogeneous.

Aequorin↗

PubMed1987

Sequence comparisons of complementary DNAs encoding aequorin isotypes.

Aequorin is the Ca2+-activated photoprotein which participates in the bioluminescence from the circumoral ring of the hydromedusa Aequorea victoria. The nucleotide sequences of five aequorin cDNAs have been compared and shown to code for three aequorin isoforms. The cDNA AEQ1 contains the entire protein coding region of 196 amino acids. The other four cDNAs contain only 70-90% of the coding region and apparently code for at least two other isoforms whose amino acid sequences differ significantly from that encoded by AEQ1. The nucleotide sequences coding for the three isotypes differ at a minimum of 54 positions out of a total of 588 nucleotides necessary to code for apoaequorin. Of these nucleotide differences, 24 account for 23 amino acid replacements, substantiating the microheterogeneity observed during sequencing of purified native aequorin [Charbonneau, H., Walsh, K.A., McCann, R.O., Prendergast, F.G., Cormier, M.J., & Vanaman, T.C. (1985) Biochemistry 24, 6762-6771]. Comparison of the deduced cDNA translations with the native protein sequences suggests the loss of seven residues from the amino terminus during purification of aequorin from Aequorea. Aequorin rapidly extracted from the jellyfish using conditions to minimize proteolysis is shown to have a larger molecular weight than that of purified native aequorin. Escherichia coli expressed aequorin encoded by AEQ1 is shown to have the same molecular weight and isoelectric point as those of one of the isotypes rapidly extracted from Aequorea.

Aequorin↗

PubMed1985

Amino acid sequence of the calcium-dependent photoprotein aequorin.

The Ca(II)-dependent photoprotein aequorin produces the luminescence of the marine coelenterate Aequorea victoria. The complete amino acid sequence of aequorin has been determined. A complete set of nonoverlapping peptides was produced by cyanogen bromide cleavage. These peptides were aligned by using the amino-terminal sequence of the intact protein and the sequences of selected arginyl and lysyl cleavage products. Although the aequorin preparations employed in these studies were homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the presence of a minimum of 3 isotypes was demonstrated by the location of 17 sites of sequence microheterogeneity. Two amino acid variants were observed at each of 16 positions while 1 position had 3 different replacements. The protein as isolated has 189 amino acids with an unblocked amino terminus. According to the sequence reported here, the molecular weight of the apoprotein is 21 459 while that of the holoprotein is 21 914. The molecule possesses three internally homologous domains which were judged to be EF-hand Ca(II) binding domains by several different criteria. Aequorin is homologous to troponin C and to calmodulin. These findings demonstrate that aequorin is a member of the Ca(II) binding protein superfamily.

Aequorin↗

PubMed1985

Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein.

Aequorin is a bioluminescent protein which consists of a polypeptide chain (apoaequorin), coelenterate luciferin, and bound oxygen. Aequorin produces blue light upon binding Ca2+. We have isolated six recombinant pBR322 plasmids which contain apoaequorin cDNA sequences. A mixed synthetic pBR322 plasmids which contain apoaequorin cDNA sequences. A mixed synthetic oligonucleotide probe was used to identify these cDNAs. An extract of an E. coli strain possessing the largest cDNA contained apoaequorin. This apoaequorin can be converted to aequorin in the presence of coelenterate luciferin, 2-mercaptoethanol, and O2. This cDNA is therefore apparently full-length.

Aequorin↗