Site-specifically labeled photoprotein-thyroxine conjugates using aequorin mutants containing unique cysteine residues: applications for binding assays (Part II).
The jellyfish Aequorea victoria produces a protein, aequorin, which belongs to the class of Ca(2+)-dependent photoproteins known for their ability to emit visible light. This property of aequorin has allowed for its as a bioluminescent label in binding assays for a variety of analytes. Due to the excellent detection limits we demonstrated in assays for small peptides using a fusion protein between the peptide of interest and the photoprotein, our next goal was to expand the range of possible analytes for producing homogeneous populations of conjugates with the aequorin label to those that were nonpeptidic in nature. Recently, we prepared and characterized four aequorin mutants containing unique cysteine residues at various positions in the polypeptide chain. In the work reported here, the four aequorin mutants were each conjugated with a maleimide-activated methyl ester derivative of thyroxine, a hormone frequently determined to evaluate thyroid function. The thyroxine-aequorin mutant conjugates were characterized in terms of the bioluminescence activities and binding properties with an anti-thyroxine monoclonal antibody for possible future employment in either heterogeneous or homogeneous binding assays for thyroxine and/or other desired analytes.
Lewis
J C
JC
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055, USA.
Cullen
L C
LC
Daunert
S
S
D000331:Aequorin / Q000737:chemistry / Q000235:genetics*; D000345:Affinity Labels / Q000737:chemistry* / Q000592:standards; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D003545:Cysteine; D007118:Immunoassay / Q000379:methods; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013974:Thyroxine / Q000031:analogs & derivatives / Q000737:chemistry* / Q000276:immunology*
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D008164:Luminescent Proteins; D000331:Aequorin; D003545:Cysteine; D013974:Thyroxine