Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia.
J Mol Recognit. 2013 Oct;26(10):450-60. doi: 10.1002/jmr.2286.
An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of three patients with systemic lupus erythematosus was used. Phage particles displaying DNA binding light chains were isolated by affinity chromatography on DNA-cellulose, and the fraction eluted by an acidic buffer (pH 2.6) was used for preparation of individual monoclonal light chains (MLChs, 28 kDa). Thirty three of 687 individual colonies obtained were randomly chosen for study of MLCh DNase activity. Nineteen of 33 clones contained MLChs with DNase activity. Four preparations of MLChs were expressed in Escherichia coli in soluble form, purified by metal chelating chromatography followed by gel filtration, and studied in detail. Detection of DNase activity after SDS-PAGE in a gel containing DNA demonstrated that the four MLChs are not contaminated by canonical DNases. The MLChs demonstrated one or two pH optima. They were inactive after the dialysis against ethylenediaminetetraacetic acid but could be activated by several externally added metal ions; the ratio of relative activity in the presence of Mg(2+) , Mn(2+) , Ni(2+) , Ca(2+) , Zn(2+) , and Co(2+) was individual for each MLCh preparation. K(+) and Na(+) inhibited the DNase activity of various MLChs at different concentrations. Hydrolysis of DNA by all four MLCh was saturable and consistent with Michaelis-Menten kinetics. These clones are the first examples of recombinant MLChs possessing high affinity for DNA (Km = 3-9 nM) and demonstrating high kcat values (3.4-6.9 min(-1) ). These observations suggest that the systemic lupus erythematosus light chain repertoire can serve as a source of new types of DNases.
利用源自三名系统性红斑狼疮患者外周血淋巴细胞的免疫球蛋白轻链噬菌粒文库，通过在 DNA-纤维素上进行亲和层析分离展示 DNA 结合轻链的噬菌体颗粒，并用酸性缓冲液（pH 2.6）洗脱，用于制备个体单克隆轻链（MLCh，28 kDa）。从 687 个单个菌落中随机选择 33 个进行 MLCh DNA 酶活性研究。33 个克隆中有 19 个含有具有 DNA 酶活性的 MLCh。以可溶形式在大肠杆菌中表达了 4 种 MLCh 制剂，通过金属螯合层析和凝胶过滤进行纯化，并进行了详细研究。在含有 DNA 的凝胶中进行 SDS-PAGE 后检测 DNA 酶活性表明，这 4 种 MLCh 未被经典 DNA 酶污染。MLCh 表现出一个或两个 pH 最佳值。经乙二胺四乙酸透析后它们无活性，但可被几种外加金属离子激活；每种 MLCh 制剂的相对活性存在时的 Mg(2+)、Mn(2+)、Ni(2+)、Ca(2+)、Zn(2+)和 Co(2+)的比值是个体的。不同浓度的 K(+)和 Na(+)抑制各种 MLCh 的 DNA 酶活性。所有 4 种 MLCh 的 DNA 水解均为饱和性，符合米氏动力学。这些克隆是具有高亲和力 DNA（Km = 3-9 nM）和高 kcat 值（3.4-6.9 min(-1)）的重组 MLCh 的第一个例子。这些观察结果表明，系统性红斑狼疮轻链库可作为新型 DNA 酶的来源。
