School of Life Sciences, University of Westminster, 115 New Cavendish St, London, W1W 6UW, UK.
BMC Biotechnol. 2011 Nov 30;11:117. doi: 10.1186/1472-6750-11-117.
Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated VH/VL interfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1.
Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either Discosoma or Aequorea in-between the variable regions of anti-p185HER2-ECD antibody 4D5-8 resulted in optimal VH/VL interface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly4Ser)3 linker precipitated at physiological pH 7.4.
This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative in situ, in vivo and ex vivo imaging, cell sorting and cell trafficking studies. Assembling the single chain antibody with monomeric fluorescent protein linker facilitates optimal variable domain pairing and alters the isoelectric point of the recombinant 4D5-8 protein conferring solubility at physiological pH 7.4. The efficient intracellular expression of these functional molecules opens up the possibility of developing an alternative approach for tagging intracellular targets with fluorescent proteins for a range of molecular cell biology imaging studies.
抗体-荧光染料缀合物是当代分子细胞生物学中用于成像、细胞分选和跟踪细胞内事件的非常宝贵的试剂。然而，在某些情况下，它们会受到批次间变异、结合部分丢失和光漂白敏感性的影响。理论上，这些问题都可以通过直接将重组抗体融合到遗传编码的荧光报告蛋白上来解决。然而，通过长柔性接头连接的单链片段可变区本身容易发生解离和聚集，并且在某些情况下，等电点与在生理相关环境中使用不兼容。在这里，我们描述了一种通用方法，该方法允许在细胞内生产一系列具有最佳 VH/VL 界面取向和等电点的彩色荧光重组抗体，这些抗体可在生理 pH 值为 7.4 的溶液中使用，并且与荧光团的结合位点比为 1:1。
在这里，我们报告了一组新型抗体荧光蛋白融合构建体的设计、组装、细菌内生产和纯化。在抗 p185HER2-ECD 抗体 4D5-8 的可变区之间插入来自 Discosoma 或 Aequorea 的单体荧光蛋白，导致最佳 VH/VL 界面相互作用，从而产生具有单个结合位点的可溶性彩色抗体，等电点为 6.5-6。用于 SK-BR-3 细胞染色研究的荧光抗体保留了荧光团性质和抗体特异性功能，而具有（Gly4Ser）3 接头的常规 4D5-8 单链抗体在生理 pH 值为 7.4 时沉淀。
这种模块化的单体重组荧光抗体平台可用于创建一系列用于定量原位、体内和体外成像、细胞分选和细胞迁移研究的重组彩色抗体分子。将单链抗体与单体荧光蛋白接头组装在一起，可促进可变区配对的最佳化，并改变重组 4D5-8 蛋白的等电点，使其在生理 pH 值为 7.4 时具有可溶性。这些功能性分子的高效细胞内表达为用荧光蛋白标记细胞内靶标开辟了可能性，用于一系列分子细胞生物学成像研究。
