Thermostable peroxidase activity with a recombinant antibody L chain-porphyrin Fe(III) complex

• Published in: FEBS letters Vol. 375; no. 3; pp. 273 - 276
• Main Authors: Takagi, Masahiro, Kohda, Katsunori, Hamuro, Takuya, Harada, Akira, Yamaguchi, Hiroyasu, Kamachi, Mikiharu, Imanaka, Tadayuki
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 20.11.1995
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - metabolism; Catalytic antibody; Cloning, Molecular; Enzyme Stability; Genes, Immunoglobulin; Haptens; Hot Temperature; Hybridomas; Immunoglobulin Light Chains - biosynthesis; Immunoglobulin Light Chains - chemistry; Immunoglobulin Light Chains - metabolism; Kinetics; L chain; Mice; Mice, Inbred BALB C - immunology; Molecular Sequence Data; Peroxidase; Peroxidases - biosynthesis; Peroxidases - chemistry; Peroxidases - metabolism; Porphyrin; Porphyrins - immunology; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Thermodynamics; Time Factors; Catalytic antibody; Peroxidase; Porphyrin; L chain
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(95)01224-3

In order to engineer a new type of catalytic antibody, we attempt to use a monoclonal antibody L chain as a host protein for a porphyrin. TCPP (meso-tetrakis(4-carboxyphenyl)porphyine) was chemically synthesized and Balb/c mice were immunized using TCPP as a hapten. Two hybridoma cells (03-1, 13-1), that produce monoclonal antibody against TCPP, were obtained. Genes for both H and L chains of monoclonal antibodies were cloned, sequenced and overexpressed using E. coli as a host. ELISA and fluorescence quenching method show that the independent antibody L chains from both Mab03-1 and Mab13-1 have specific interaction with TCPP. Furthermore, the recombinant antibody L chain from Mab13-1 exhibits much higher peroxidase activity than TCPP Fe(III) alone. The enzyme activity was detectable with pyrogallol and ABTS (2,2-azinobis-3-ethylbenzthiazolin-6-sulfonic acid) but not with catechol. This new catalytic antibody was extremely thermostable. Optimum temperature of the peroxidase reaction by the complex of 13-1L chain and TCPP Fe(III) was 90°C, while that the TCPP Fe(III) alone was 60°C.