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.
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada N9B 3P4.
Monique
M
Verhaegen
Theodore K
TK
Christopoulos
D000331:Aequorin / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000235:genetics; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012097:Repressor Proteins; D013194:Staining and Labeling; D014157:Transcription Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D000331:Aequorin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli