A fusion protein of the synthetic IgG-binding domain and aequorin: Expression and purification from E. coli cells and its application

- The fusion protein of ZZ domain and apoaequorin was expressed in E. coli cells.
- The fusion protein was highly purified using dual-affinity chromatography.
- Regenerated ZZ-aequorin exhibited similar luminescence properties to native aequorin.
- ZZ-aequorin could be used as a reporter for detecting IgG (antibodies).

Aequorin is a Ca2+-binding photoprotein that is a complex of apoaequorin (apoAQ) and 2-peroxycoelenterazine. In this study, the fusion protein (ZZ-apoAQ) composed of the synthetic IgG-binding domain (ZZ domain) derived from Staphylococcus aureus protein A and apoAQ was expressed into the periplasmic space of Escherichia coli cells. ZZ-apoAQ was highly purified using Ni-chelate affinity chromatography followed by IgG affinity chromatography. ZZ-AQ was prepared from purified ZZ-apoAQ by incubation with coelenterazine and was characterized, including its luminescence properties. ZZ-AQ could be used as a reporter for detecting IgG and the measurable range of IgG coated on a 96-well plate was 1-1000 ng/mL.