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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Cleavage of supercoiled plasmid DNA by autoantibody Fab fragment: application of the flow linear dichroism technique.
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Cleavage of supercoiled plasmid DNA by autoantibody Fab fragment: application of the flow linear dichroism technique.

机译：自身抗体Fab片段切割超螺旋质粒DNA：流线性二色性技术的应用。

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摘要

A highly effective method consisting of two affinity chromatography steps and ion-exchange and gel-filtration chromatography steps was developed for purification of autoantibodies from human sera with DNA-hydrolyzing activity. Antibody Fab fragment, which had been purified 130-fold, was shown to catalyze plasmid DNA cleavage. The flow linear dichroism technique was used for quantitative and qualitative studying of supercoiled plasmid DNA cleavage by these autoantibodies in comparison with DNase I and EcoRI restriction endonuclease. The DNA autoantibody Fab fragment was shown to hydrolyze plasmid DNA by Mg(2+)-dependent single-strand multiple nicking of the substrate. Kinetic properties of the DNA autoantibody Fab fragment were evaluated from the flow linear dichroism and agarose gel electrophoresis data and revealed a high affinity (Kobsm = 43 nM) and considerable catalytic efficiency (kappcat/Kobsm = 0.32 min-1.nM-1) of the reaction.
机译：开发了一种由两个亲和色谱步骤以及离子交换和凝胶过滤色谱步骤组成的高效方法，用于从具有DNA水解活性的人血清中纯化自身抗体。已显示已纯化130倍的抗体Fab片段可催化质粒DNA切割。与DNase I和EcoRI限制性核酸内切酶相比，流动线性二色性技术用于这些自体抗体对超螺旋质粒DNA切割的定量和定性研究。 DNA自身抗体Fab片段显示可通过Mg（2+）依赖性底物的单链多切口水解质粒DNA。从流动线性二色性和琼脂糖凝胶电泳数据评估了DNA自身抗体Fab片段的动力学特性，并显示出高亲和力（Kobsm = 43 nM）和相当高的催化效率（kappcat / Kobsm = 0.32 min-1.nM-1）。反应。

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