CONSTRUCTION OF CATECHOL 2,3-DIOXYGENASE- PROTEIN-G CHIMERIC PROTEINSAND ITS APPLICATION TO IMMUNOASSAY

To make a useful reagent for the immunoassay, chimeric proteins of a c atechol 2,3-dioxygenase (C 23 O), which converts catechol to a yellow compound with absorption maximum at 375 nm, and an IgG binding domain of protein G from Streptococcus sp. were designed and expressed in Esc herichia coli. A cloned gene for C 23 O from Pseudomonas aeruginosa JI 104 and a synthesized protein G C 1 domain gene, were fused and expre ssed under lac promoter derived from pTV 118 N plasmid. When 5 or 9 am ino acids linkers were inserted between two domains, functional chimer ic proteins with C 23 O activity and human IgG-binding ability were ob tained. The chimeric protein with 9 amino acids linker was further tes ted for its utility in enzyme-linked immunosorbent assay (ELISA) to de tect bovine serum albumin (BSA). Microgram per millilitter order of BS A was reproducibly detected in ELISA using a 96 well microtiter plate and a frequently used microtiter plates reader with a 410 nm filter.