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Thermostable peroxidase activity with a recombinant antibody L chain‐porphyrin Fe(III) complex
Author(s) -
Takagi Masahiro,
Kohda Katsunori,
Hamuro Takuya,
Harada Akira,
Yamaguchi Hiroyasu,
Kamachi Mikiharu,
Imanaka Tadayuki
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)01224-3
Subject(s) - pyrogallol , peroxidase , monoclonal antibody , chemistry , recombinant dna , hapten , microbiology and biotechnology , linker , antibody , porphyrin , immunoglobulin light chain , biochemistry , enzyme , biology , gene , computer science , immunology , operating system
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.