Expression and secretion of aequorin as a chimeric antibody by means of a mammalian expression vector.
A fusion protein has been expressed from the relevant genes in mammalian cells consisting of the photoprotein aequorin and an anti-4-hydroxy-3-nitrophenacetyl antibody gene. This chimeric antibody has allowed the development of a sensitive luminescent immunoassay. Initially the cDNA of the photoprotein aequorin from Aequorea victoria was cloned and expressed in Escherichia coli. The gene was expressed as apoaequorin and, by using luciferin isolated from Renilla reniformis, its activity was found essentially identical to native aequorin. The aequorin gene was subcloned into a mammalian expression vector to produce a fusion protein directing secretion of apoaequorin; the aequorin gene was fused to the 3' terminus of an immunoglobulin heavy-chain gene that directed expression of an anti-4-hydroxy-3-nitrophenacetyl antibody. The gene fusion contained the variable region, the constant region domain 1, and part of domain 2 for the IgG2b mouse immunoglobulin, followed by the aequorin gene. Transfection of the chimeric gene into a cell line expressing the complementary lambda 1 light chain, J558L, allowed recovery of a chimeric antibody with binding specificity for the 4-hydroxy-3-nitrophenacetyl group and the related 4-hydroxy-3-iodo-5-nitrophenacetyl hapten. The Ca2(+)-dependent bioluminescent activity of aequorin was also recovered.
Casadei
J
J
IGEN Inc., Rockville, MD 20852.
Powell
M J
MJ
Kenten
J H
JH
D000331:Aequorin / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D000906:Antibodies / Q000235:genetics*; D001483:Base Sequence; D002678:Chimera; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D008164:Luminescent Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D015183:Restriction Mapping; D037401:Scyphozoa
D000906:Antibodies; D008164:Luminescent Proteins; D015345:Oligonucleotide Probes; D000331:Aequorin