Djender Selma, Schneider Aurelie, Beugnet Anne, Crepin Ronan, Desrumeaux Klervi Even, Romani Chiara, Moutel Sandrine, Perez Franck, de Marco Ario
Microb Cell Fact. 2014 Sep 16;13:140. doi: 10.1186/s12934-014-0140-1.
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.
从展示文库中分离重组抗体片段是一种强大的替代方法，可替代使用杂交瘤技术产生IgG。然后，所选抗体片段可以很容易地工程化为具有可变质量和复杂性的（多）标记构建体，并且当与Fc结构域融合表达时可重构为骆驼科IgG样分子。然而，所有抗体构建体都依赖于氧化环境来进行正确折叠，因此仍然属于难以在细菌中表达的蛋白质。在这类生物体中，它们大多在周质空间中低产量产生。
我们证明，重组抗体与多个标签的融合构建体可以在表达巯基氧化酶的细菌细胞质中高产率生产且完全具有功能。该方法应用于结构要求苛刻的分子，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了验证。
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，用于快速且廉价地生产功能性骆驼科IgG样单克隆抗体和含多个二硫键且适用于基础研究和临床应用的基于抗体的试剂。
