C-termimal and N-terminal fusions of aequorin with small peptides in immunoassay development

Aequorin fusion proteins have been used extensively in intracellular Ca2+ m easurements and in the development of binding assays. Gene fusions to aequo rin for production of fusion proteins have been so far limited to its N-ter minus, 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 aequori n through genetic engineering. The octapeptide was also fused to the N-term inus of the aequorin-leu-enkephalin fusion protein, which enables its affin ity purification. Contrary to reports of earlier studies, we found that aeq uorin retains its bioluminescence activity after modification of the C-term inus. The half-life of light emission and the calibration curves obtained w ith the fusion proteins were comparable to those of the cysteine-free mutan t of aequorin. Dose-response curves for the octapeptide were generated usin g two aequorin-octapeptide fusion proteins with the octapeptide fused to th e N-terminus in one case, and to the C-terminus in the other. Similar detec tion limits for the octapeptide were obtained using both fusion proteins. T he C-terminal fusion system has advantages in cases where antibodies recogn ize only the C-terminus of the peptide, as well as in cases where the funct ionality of the peptide lies in its C-terminus. The purification is also si mplified as the affinity tag can be engineered at one terminus and the pept ide of interest at the other.