Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies.
Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies.
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细菌细胞质作为产生基于功能VHH的亲和力试剂和Camelidae IgG类样重组抗体的有效细胞室。
DOI：
10.1186/s12934-014-0140-1
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发表时间：
2014-09-16
影响因子：
6.4
通讯作者：
de Marco A
中科院分区：
文献类型：
--
作者：
Djender S;Schneider A;Beugnet A;Crepin R;Desrumeaux KE;Romani C;Moutel S;Perez F;de Marco A
从展示的文库中分离重组抗体片段代表了使用杂交瘤技术产生IgG的有力替代方案。然后可以将所选择的抗体片段容易地工程化为可变质量和复杂性的（多）标记构建体，以及当表达融合至Fc结构域时重构为骆驼科IgG样分子。然而，所有抗体构建体都依赖于氧化环境来正确折叠，因此仍然属于难以在细菌中表达的蛋白质。在这样的生物体中，它们大多在周质空间中以低产量产生。我们证明了重组抗体与多个标签组合的融合构建体可以以高产率产生，并且在表达巯基氧化酶的细菌的细胞质中完全起作用。将该方法应用于结构要求高的分子，如与SNAP和Fc结构域融合的VHH，并在多种免疫技术（FACS、ELISA、WB、IP、SPR和IF）中使用抗体衍生试剂进行验证。所收集的数据证明了一种方法的可行性，该方法建立了一种全新的方法，用于快速和廉价地生产功能性骆驼科IgG样单克隆抗体和含有多个二硫键的基于抗体的试剂，并且适用于基础研究和临床应用。本文的在线版本（doi：10.1186/s12934-014-0140-1）包含补充材料，可供授权用户使用。
The isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. The selected antibody fragments can then be easily engineered into (multi)-tagged constructs of variable mass and complexity as well as reconstituted into Camelidae IgG-like molecules when expressed fused to Fc domains. Nevertheless, all antibody constructs depend on an oxidizing environment for correct folding and consequently still belong to the proteins difficult to express in bacteria. In such organisms they are mostly produced at low yields in the periplasmic space. We demonstrate that fusion constructs of recombinant antibodies in combination with multiple tags can be produced at high yields and totally functional in the cytoplasm of bacteria expressing sulfhydryl oxidase. The method was applied to structurally demanding molecules such as VHHs fused to SNAP and Fc domains and was validated using the antibody-derived reagents in a variety of immune techniques (FACS, ELISA, WB, IP, SPR, and IF). The collected data demonstrate the feasibility of a method that establishes a totally new approach for producing rapidly and inexpensively functional Camelidae IgG-like monoclonal antibodies and antibody-based reagents containing multiple disulfide bonds and suitable for both basic research and clinical applications. The online version of this article (doi:10.1186/s12934-014-0140-1) contains supplementary material, which is available to authorized users.
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