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Selection of catalytic antibodies for a biosynthetic reaction from a combinatorial cDNA library by complementation of an auxotrophic Escherichia coli: antibodies for orotate decarboxylation.

机译:通过营养缺陷型大肠杆菌的互补从组合cDNA文库中选择用于生物合成反应的催化抗体:用于乳清酸脱羧的抗体。

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摘要

Antibodies capable of decarboxylating orotate were sought by immunization with a hapten designed to elicit antibodies with combining sites that resemble the orotate-binding and catalytic portion of the active site of the enzyme orotidine 5'-monophosphate (OMP) decarboxylase (orotidine-5'-monophosphate carboxy-lyase, EC 4.1.1.23). Active recombinant antibody fragments (Fabs) were selected from a combinatorial cDNA library by complementation of a pyrF strain of Escherichia coli and growth of the library-expressing cells on pyrimidine-free medium. In this biological screen, a sufficiently active antibody from the library would decarboxylate orotate to produce uracil, a pyrimidine source for the auxotroph, and would provide the cells with a growth advantage compared to cells without an active antibody. Six recombinant Fabs yielded identifiable colonies in a screen of 16,000 transformants. To enhance its stability and expression level, one of the six positive fragments was converted into single-chain form. In this form, the antibody fragment conferred a definite growth advantage to the auxotroph that was eliminated when the hapten was included in the medium. The purified single-chain antibody displayed orotate decarboxylase activity in vitro, as determined by a 14CO2 displacement assay. The specific activity of the antibody is approximately 10(-7) times that of naturally occurring OMP decarboxylase, but this antibody-catalyzed rate is estimated to be 10(8) times the background rate. The results offer the potential to use these methods to obtain catalytic antibodies for other biosynthetic reactions as well as to assess the effectiveness of the hapten transition state or active site analog in eliciting antibody catalysts.
机译:通过用半抗原进行免疫来寻找能够使乳清蛋白脱羧的抗体,该半抗原被设计为引发抗体,其结合位点类似于乳清苷5'-单磷酸酯(OMP)脱羧酶的活性部位的乳清酸酯结合和催化部分(orotidine-5'-一磷酸羧裂合酶,EC 4.1.1.23)。通过互补大肠杆菌的pyrF菌株并在无嘧啶的培养基上培养文库表达细胞,从组合cDNA文库中选择了活性重组抗体片段(Fabs)。在该生物学筛选中,来自文库的足够活性的抗体将使乳清蛋白脱羧以产生尿嘧啶,尿嘧啶是营养缺陷型的嘧啶源,并且与没有活性抗体的细胞相比,将为细胞提供生长优势。在16,000个转化子的筛选中,六个重组Fab产生了可识别的菌落。为了增强其稳定性和表达水平,六个阳性片段之一被转化为单链形式。以这种形式,抗体片段为营养缺陷型赋予了一定的生长优势,而当半抗原被包含在培养基中时,营养缺陷型被消除了。纯化的单链抗体在体外表现出乳清酸脱羧酶活性,这是通过14CO2置换分析确定的。抗体的比活性约为天然OMP脱羧酶的比活性的10(-7)倍,但该抗体催化的速率估计为背景速率的10(8)倍。结果提供了使用这些方法获得用于其他生物合成反应的催化抗体以及评估半抗原过渡态或活性位点类似物在引发抗体催化剂中的有效性的潜力。

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  • 作者

    Smiley, J A; Benkovic, S J;

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  • 年度 1994
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  • 原文格式 PDF
  • 正文语种 en
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