Philibert Pascal, Stoessel Audrey, Wang Wei, Sibler Annie-Paule, Bec Nicole, Larroque Christian, Saven Jeffery G, Courtête Jérôme, Weiss Etienne, Martineau Pierre
CNRS, UMR5160, CRLC, 15, av, Charles Flahault, BP14491, 34093, Montpellier Cedex 5, France.
BMC Biotechnol. 2007 Nov 22;7:81. doi: 10.1186/1472-6750-7-81.
Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.
We describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the alphabeta tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressed in vivo in human cells since they were essentially localized in the nucleus.
This new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications.
胞内抗体被定义为在细胞内异位表达的抗体分子。这类胞内抗体可用于在活细胞中可视化或抑制靶向抗原。然而，大多数抗体片段不能用作胞内抗体，因为它们在细胞质和细胞核的还原条件下无法折叠。
我们描述了一个基于独特框架构建并经过验证的大型合成人单链抗体片段文库，该文库针对细胞质表达进行了优化。通过模拟互补决定区3（CDR3）环的自然多样性对文库进行筛选，确保了单链抗体（scFv）是完全人源且具有功能的。我们表明该文库具有高度多样性和功能性，因为通过噬菌体展示能够从本研究中测试的五种蛋白质（脾酪氨酸激酶和极光激酶A、αβ微管蛋白二聚体、乳头瘤病毒E6蛋白和核心组蛋白）中分离出几种强结合剂。一些筛选出的scFv在细菌细胞质中以极高水平表达，仅从20毫升培养物中就能纯化出1毫克活性scFv。最后，我们表明在针对核心组蛋白进行三轮筛选后，超过一半的筛选出的scFv在人细胞中体内表达时具有活性，因为它们主要定位于细胞核。
这个新文库不仅是一个用于轻松大规模筛选功能性胞内抗体的有前途的工具，也是用于分离可用于众多生物技术和治疗应用的高表达scFv的工具。
