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.
Stults
N L
NL
Department of Biochemistry, University of Georgia, Athens 30602.
Stocks
N F
NF
Rivera
H
H
Gray
J
J
McCann
R O
RO
O'Kane
D
D
Cummings
R D
RD
Cormier
M J
MJ
Smith
D F
DF
D000331:Aequorin / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D001710:Biotin / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics*; D005577:Forssman Antigen / Q000032:analysis; D005822:Genetic Vectors; D005915:Globosides / Q000032:analysis; D008163:Luminescent Measurements; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D037401:Scyphozoa
D001059:Apoproteins; D002352:Carrier Proteins; D005915:Globosides; D008565:Membrane Proteins; D011994:Recombinant Proteins; C060014:apoaequorin; C017071:biotin-binding proteins; D000331:Aequorin; C010163:Forssman glycolipid; D001710:Biotin; D005577:Forssman Antigen