Use of recombinant biotinylated aequorin in microtiter and membrane-based assays: Purification of recombinant apoaequorin from Escherichia coli
| Autor: | Hilda N. Rivera, Judy Gray, Richard O. McCann, David F. Smith, Milton J. Cormier, Dennis O'Kane, Nancy L. Stults, Richard D. Cummings, Nancy F. Stocks |
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| Rok vydání: | 1992 |
| Předmět: |
Streptavidin
Scyphozoa Genetic Vectors Aequorin Photoprotein Biotin Biochemistry law.invention chemistry.chemical_compound Drug Stability law Escherichia coli Animals Bioluminescence Forssman Antigen Globosides biology Membrane Proteins biology.organism_classification Luciferin Recombinant Proteins chemistry Biotinylation Luminescent Measurements biology.protein Recombinant DNA Aequorea victoria Apoproteins Carrier Proteins |
| Zdroj: | Biochemistry. 31:1433-1442 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00120a021 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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