RiNA Netzwerk RNA-Technologien GmbH, Berlin, Germany.
Biotechniques. 2012 Sep;53(3):153-60. doi: 10.2144/0000113904.
A eukaryotic cell-free system based on Spodoptera frugiperda cells was developed for the convenient synthesis of Fab antibody fragments and other disulfide bridge containing proteins. The system uses (i) a cell lysate that is mildly prepared under slightly reduced conditions, thus maintaining the activity of vesicles derived from the endoplasmic reticulum, (ii) signal peptide dependent translocation into these vesicles, and (iii) a redox potential based on reduced and oxidized glutathione. Monomeric heavy and light immunoglobulin chains are almost completely converted to highly active dimeric Fab joined by intermolecular disulfide bridges without supplementation of chaperones or protein disulfide isomerase. The applicability of the system is demonstrated by the synthesis of anti-lysozyme and anti-CD4 Fab antibody fragments yielding approximately 10 µg Fab per milliliter reaction mixture. The lack of endotoxins in this system is a prerequisite that synthesized Fab can be applied directly using whole synthesis reactions in cell-based assays that are sensitive to this substance class. Moreover, the system is compatible with PCR-generated linear templates enabling automated generation of antibody fragments in a high-throughput manner, and facilitating its application for screening and validation purposes.
基于 S. frugiperda 细胞的真核无细胞体系被开发出来，用于方便地合成 Fab 抗体片段和其他含有二硫键的蛋白质。该体系使用：（i）在轻微还原条件下温和制备的细胞裂解物，从而保持内质网衍生小泡的活性；（ii）依赖信号肽的易位进入这些小泡；（iii）基于还原型和氧化型谷胱甘肽的氧化还原电势。单体重链和轻链免疫球蛋白几乎完全转化为具有高活性的二聚体 Fab，通过分子间二硫键连接，无需补充伴侣蛋白或蛋白质二硫键异构酶。该体系的适用性通过合成抗溶菌酶和抗 CD4 Fab 抗体片段得到证明，每毫升反应混合物可产生约 10 µg Fab。该体系中不存在内毒素是一个前提条件，即合成的 Fab 可以直接用于基于细胞的测定中的全合成反应，这些测定对这类物质很敏感。此外，该体系与 PCR 生成的线性模板兼容，能够以高通量的方式自动生成抗体片段，并促进其在筛选和验证目的中的应用。
