Abstracts

ZZ-AmyLuc is a fusion protein consisting of the ZZ-domain of protein A and Amydetes vivianii (Amy) firefly luciferase, which was designed for use in bioluminescent immunoassays and Western Blotting. ZZ-AmyLuc offers a more sensitive and stable alternative to chemiluminescence methods based on HPR for detecting primary antibodies against specific antigens, including anti-SARS-COV-2 nucleoprotein. The goal of this study was to characterize the physical-chemical properties of ZZ-AmyLuc, and to improve its sensitivity and stability for bioluminescent immunoassays. For this purpose, E. coli BL-21 bacteria transformed with pCold-ZZ-AmyLuc cDNA were used for heterologous expression, and ZZ-AmyLuc was extracted and purified by Nickel-agarose affinity chromatography. The kinetics and spectra properties of the purified fusion protein were characterized and the immunoassay conditions optimized. The fusion protein had an optimum pH ~ 9.0, which is higher than that of the Wild-type AmyLuc (WT). The KM for ATP (22 µM) and luciferin (30 µM) increased in relation to the wild-type Amy-Luc (WT), corroborating the slower and more sustained luminescent kinetics of the phusion protein, which can be advantageous for immunoassays. Furthermore, ZZ-AmyLuc showed a kcat of 1.5 . 10-4 cps), slightly higher than the value found for WT (1.09 . 10-4 cps), but its catalytic efficiency was slightly lower (6.8 c.s-1.µM-1 for ATP and 5.0 c.s-1.µM-1 for luciferin) when compared to WT luciferase (12.1 c.s-1.µM-1 for ATP and luciferin). The fusion protein BL spectrum overlapped with that of the WT luciferase (λmax=549 nm), and showed similar pH-sensitivity. The immunoassays in the current format detected up to 0.5 ng of SARS-CoV-2 nucleoprotein, highlighting the need for further studies to increase the detection limit, such as engineering this fusion protein and changing the composition of the assay solution. Thus, ZZ-AmyLuc fusion protein shows great potential to be used as an efficient and sensitive alternative for large-scale IgG-based immunoassays and Western Blotting.