Zeng J, Li H C, Tanaka T, Rabbitts T H
Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
J Immunol Methods. 2015 Nov;426:140-3. doi: 10.1016/j.jim.2015.08.009. Epub 2015 Aug 25.
Protein functions that are mediated by interaction with other proteins (protein-protein interactions, PPI) are important for normal cell biology and also in disease. Molecules that can interfere with PPI are required as laboratory tools to dissect function, as lead drug surrogates for target validation and as templates for drug discovery. We describe enhanced developments to Intracellular Antibody Capture (IAC) technology that can select antibody fragments able to interact with targets in cells. This is illustrated by the isolation of single heavy chain variable region domains binding to the basic-helix-loop-helix and leucine zipper region of the CMYC oncogenic protein. The enhanced IAC (eIAC) methodology deploys screening in yeast cells of a single diverse library initially with randomization only of CDR3. Further sequential randomization of CDR2 and CDR1 of three independently selected anti-CMYC clones illustrates an in vivo affinity maturation process. This concise eIAC approach facilitates the rapid selection of antibody fragments to explore the proteome interaction spectrum of mammalian cells and disease targeting.
通过与其他蛋白质相互作用介导的蛋白质功能（蛋白质-蛋白质相互作用，PPI）对于正常细胞生物学以及疾病过程都很重要。能够干扰PPI的分子作为剖析功能的实验室工具、作为用于靶点验证的先导药物替代物以及作为药物发现的模板是必不可少的。我们描述了对细胞内抗体捕获（IAC）技术的改进，该技术能够筛选出能够在细胞内与靶标相互作用的抗体片段。这通过分离与CMYC致癌蛋白的碱性螺旋-环-螺旋和亮氨酸拉链区域结合的单重链可变区结构域得到了证明。改进后的IAC（eIAC）方法首先在酵母细胞中对单一多样化文库进行筛选，最初仅对互补决定区3（CDR3）进行随机化。对三个独立选择的抗CMYC克隆的互补决定区2（CDR2）和互补决定区1（CDR1）进一步进行顺序随机化，展示了一种体内亲和力成熟过程。这种简洁的eIAC方法有助于快速筛选抗体片段，以探索哺乳动物细胞的蛋白质组相互作用谱和疾病靶向。
