Thermostable peroxidase activity with a recombinant antibody L chain-porphyrin Fe(III) complex.
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 degrees C, while that the TCPP Fe(III) alone was 60 degrees C.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005803:Genes, Immunoglobulin; D006241:Haptens; D006358:Hot Temperature; D006825:Hybridomas; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011166:Porphyrins / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D013997:Time Factors
D000911:Antibodies, Monoclonal; D006241:Haptens; D007147:Immunoglobulin Light Chains; D011166:Porphyrins; D011994:Recombinant Proteins; C018395:tetracarboxyphenylporphine; D010544:Peroxidases