Takagi M, Kohda K, Hamuro T, Harada A, Yamaguchi H, Kamachi M, Imanaka T
Department of Biotechnology, Faculty of Engineering, Osaka University, Japan.
FEBS Lett. 1995 Nov 20;375(3):273-6. 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 degrees C, while that the TCPP Fe(III) alone was 60 degrees C.
为了构建一种新型催化抗体，我们尝试使用单克隆抗体轻链作为卟啉的宿主蛋白。化学合成了四（对羧基苯基）卟啉（TCPP），并以TCPP作为半抗原免疫Balb/c小鼠。获得了两个产生抗TCPP单克隆抗体的杂交瘤细胞（03-1、13-1）。克隆了单克隆抗体重链和轻链的基因，进行测序并以大肠杆菌作为宿主进行过表达。酶联免疫吸附测定（ELISA）和荧光猝灭法表明，来自Mab03-1和Mab13-1的独立抗体轻链均与TCPP有特异性相互作用。此外，来自Mab13-1的重组抗体轻链比单独的TCPP Fe（III）表现出更高的过氧化物酶活性。用连苯三酚和2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸（ABTS）可检测到酶活性，但用儿茶酚则检测不到。这种新型催化抗体具有极高的热稳定性。13-1轻链与TCPP Fe（III）复合物的过氧化物酶反应的最佳温度为90℃，而单独的TCPP Fe（III）的最佳温度为60℃。
