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

Abstract

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.

Bioluminescence Immunoassay for Cortisol Using Recombinant Aequorin as a Label

“…The relative bioluminescent activity of each cortisol-AEQ mutant S conjugate was calculated with respect to AEQ mutant S. The results, reported in Table 1, show that the increasing molar excess of cortisol to AEQ mutant S caused a decrease in the bioluminescence activity. A similar effect of gradual loss of activity of aequorin as a consequence of increasing conjugation ratio to a small analyte has been previously reported (22). Nevertheless, the 500:1 conjugate still retained 28% of the bioluminescent activity.…”

Hybrid Minimal Core Streptavidin–Obelin as a Versatile Reporter for Bioluminescence‐based Bioassay

“…In spite of the fact that obelin and other photoproteins are relatively stable to chemical modifications (the loss of bioluminescence activity does not exceed 20–40% ) the partial inactivation and unpredictable heterogeneity of the conjugates can negatively affect specificity and sensitivity of the assays. Genetic engineering provides the possibility of fusing photoprotein to biospecific molecules which will be homogeneous, with 1:1 stoichiometry and a high activity of photoprotein moiety.…”

Evidence ForDe NovoBiosynthesis of Coelenterazine in the Bioluminescent Midwater Shrimp,Systellaspis DebilisC

“…Three hauls, each at a different station position, were taken with an 8-m 2 mouth-area rectangular midwater trawl. The bacteria Escherichia coli BL21(DE3) were induced to produce high levels of apo-aequorin which was subsequently purified on a Sephacryl (S100) column at 4°C, then pooled and stored at -70°C (Stults et al, 1992;Watkins, unpublished data). The number of eggs were counted (ranging from 8 to 21 eggs), their development noted and the coelenterazine content of the whole clutch measured immediately.…”

“…Bulk preparations of recombinant apo-aequorin were prepared using the T7 RNA polymerase driven expression system, pET (Studier et al, 1990). The bacteria Escherichia coli BL21(DE3) were induced to produce high levels of apo-aequorin which was subsequently purified on a Sephacryl (S100) column at 4°C, then pooled and stored at -70°C (Stults et al, 1992;Watkins, unpublished data).…”