Abstract
To make a useful reagent for the immunoassay, chimeric proteins of a catechol 2, 3-dioxygenase (C23O), 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 Escherichia coli. A cloned gene for C23O from Pseudomonas aeruginosa JI 104 and a synthesized protein GC1 domain gene, were fused and expressed under lac promoter derived from pTV118N plasmid. When 5 or 9 amino acids linkers were inserted between two domains, functional chimeric proteins with C23O activity and human IgG-binding ability were obtained. The chimeric protein with 9 amino acids linker was further tested for its utility in enzyme-linked immunosorbent assay (ELISA) to detect bovine serum albumin (BSA). Microgram per millilitter order of BSA was reproducibly detected in ELISA using a 96 well microtiter plate and a frequently used microtiter plates reader with a 410 nm filter.
