Fraunhofer Institute for Biomedical Engineering, Branch Potsdam-Golm, Potsdam, Germany.
J Biotechnol. 2012 Dec 15;164(2):220-31. doi: 10.1016/j.jbiotec.2012.08.020. Epub 2012 Sep 7.
Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner.
无细胞蛋白质合成越来越受到关注，因为它可以快速、高通量地合成许多蛋白质，特别是抗体片段。在这项研究中，我们提出了一种在真核体外翻译系统中生产单链抗体片段（scFv）的新策略。该策略包括在两种不同制备的昆虫细胞裂解物中进行模型抗体片段的无细胞表达、分离和无标记相互作用分析。这些裂解物包含来自内质网（ER）的具有转位活性的微粒体结构，允许对无细胞合成的蛋白质进行翻译后修饰。这两种昆虫细胞裂解物都能够将 scFv 合成并转位到 ER 衍生的囊泡中。然而，只有具有特定适应的氧化还原电势的裂解物才能产生功能活性的抗体片段。我们开发了一种新的基于 scFv 无细胞产物转位到微粒体结构和随后收集富含蛋白质的囊泡的方法，用于分离功能目标蛋白。从这些囊泡中释放的抗体片段非常适合用于无标记结合研究。总之，这些结果表明昆虫细胞裂解物具有在易于处理和节省时间的方式下生产、纯化和选择抗体片段的潜力。
