C-terminal and n-terminal fusions of aequorin with small peptides in immunoassay development.
Aequorin fusion proteins have been used extensively in intracellular Ca2+ measurements and in the development of binding assays. Gene fusions to aequorin for production of fusion proteins have been so far limited to its N-terminus, as previous studies have indicated that aequorin loses its activity upon modification of its C-terminus. To further investigate this, two model peptides, an octapeptide (DTLDDDDL), and leu-enkephalin (TGGFL), an opioid peptide, were fused to the C-terminus of a cysteine-free mutant of aequorin through genetic engineering. The octapeptide was also fused to the N-terminus of the aequorin-leu-enkephalin fusion protein, which enables its affinity purification. Contrary to reports of earlier studies, we found that aequorin retains its bioluminescence activity after modification of the C-terminus. The half-life of light emission and the calibration curves obtained with the fusion proteins were comparable to those of the cysteine-free mutant of aequorin. Dose-response curves for the octapeptide were generated using two aequorin-octapeptide fusion proteins with the octapeptide fused to the N-terminus in one case, and to the C-terminus in the other. Similar detection limits for the octapeptide were obtained using both fusion proteins. The C-terminal fusion system has advantages in cases where antibodies recognize only the C-terminus of the peptide, as well as in cases where the functionality of the peptide lies in its C-terminus. The purification is also simplified as the affinity tag can be engineered at one terminus and the peptide of interest at the other.
Deo
S K
SK
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055, USA.
Lewis
J C
JC
Daunert
S
S
D000331:Aequorin / Q000235:genetics; D002138:Calibration; D004305:Dose-Response Relationship, Drug; D004743:Enkephalin, Leucine / Q000235:genetics; D005818:Genetic Engineering; D007118:Immunoassay / Q000379:methods / Q000592:standards; D008163:Luminescent Measurements; D009842:Oligopeptides / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000235:genetics; D012680:Sensitivity and Specificity
D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000331:Aequorin; D004743:Enkephalin, Leucine