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首页> 外文期刊>Antibodies >The Biotechnological Applications of Recombinant Single-Domain Antibodies are Optimized by the C-Terminal Fusion to the EPEA Sequence (C Tag)
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The Biotechnological Applications of Recombinant Single-Domain Antibodies are Optimized by the C-Terminal Fusion to the EPEA Sequence (C Tag)

机译：重组单域抗体的生物技术应用是通过C末端与EPEA序列（C标签）的融合而优化的。

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摘要

We designed a vector for the bacterial expression of recombinant antibodies fused to a double tag composed of 6xHis and the EPEA amino acid sequence. EPEA sequence (C tag) is tightly bound by a commercial antibody when expressed at the C-term end of a polypeptide. The antigen is released in the presence of 2 M MgCl2. Consequently, constructs fused to the 6xHis-C tags can be purified by two successive and orthogonal affinity steps. Single-domain antibodies were produced either in the periplasmic or in the cytoplasmic space of E. coli. Surprisingly, the first affinity purification step performed using the EPEA-binding resin already yielded homogeneous proteins. The presence of the C tag did not interfere with the binding activity of the antibodies, as assessed by FACS and SPR analyses, and the C tag was extremely effective for immunoprecipitating HER2 receptor. Finally, the Alexa488-coupled anti-C tag allowed for simplification of FACS and IF analyses. These results show that a tag of minimal dimensions can be effectively used to improve the applicability of recombinant antibodies as reagents. In our hands, C tag was superior to His-tag in affinity purification and pull-down experiments, and practical in any other standard immune technique.
机译：我们设计了一种用于细菌表达与6xHis和EPEA氨基酸序列组成的双标签融合的重组抗体的载体。当在多肽的C末端表达时，EPEA序列（C标签）与商业抗体紧密结合。抗原在2 M MgCl 2 存在下释放。因此，可以通过两个连续且正交的亲和步骤纯化与6xHis-C标签融合的构建体。在大肠杆菌的周质或细胞质空间中产生单结构域抗体。出人意料的是，使用EPEA结合树脂进行的第一个亲和纯化步骤已经产生了均质蛋白。通过FACS和SPR分析评估，C标签的存在不会干扰抗体的结合活性，并且C标签对于免疫沉淀HER2受体非常有效。最后，Alexa488偶联的抗C标签可简化FACS和IF分析。这些结果表明，最小尺寸的标签可以有效地用于改善重组抗体作为试剂的适用性。在我们手中，C标记物在亲和纯化和下拉实验中优于His标记物，并且在任何其他标准免疫技术中都是实用的。

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