Abstract
E.coli alkaline phosphatase (PhoA) is a thermally stable homodimeric metalloenzyme with resistance to many proteases. In previous studies, several loops in PhoA were known to permit peptide insertion without destroying its structure as well as the enzymatic activity. Especially, an epitope insertion near the active site (407-408) allowed modulation of enzymatic activity upon binding with corresponding antibody. Based on this observation, we decided to make a novel 'allosteric' enzyme that can accommodate desired receptor domains, whose activity can be controlled by the binding of its ligand as a molecular biosensor. To this end, construction of circularly permutated PhoAs having new N- and C-terminus at permissive loops (91-93/407-408) near the active center was attempted, with the original termini connected by (G4S)3 peptide linker. When the DNA encoding these circular permutants were inserted to pET20b plasmid and expressed in E. coli BL21(DE3,pLysS), an active enzyme was obtained only for the former having new C-terminus at aa 90 of the wild-type PhoA (cp90). Then an epitope of osteocalcin, an important marker of bone metabolism, was inserted to the new N-terminus of cp90, with its original 91 and 93th residues randomized to obtain optimal variants. Interestingly, some resultant fusion proteins showed similar specific activity to that of wild-type PhoA, and showed significant reduction in Vmax/Km in the presence of anti-osteocalcin antibody. The result indicates that the epitope-tagged cp90 can act as a molecular sensor to detect specific antibodies, which will enable competitive detection of specific antigen epitopes
