Birnboim-Perach Racheli, Grinberg Yehudit, Vaks Lilach, Nahary Limor, Benhar Itai
School of Molecular Cell Biology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, Israel.
Methods Mol Biol. 2019;1904:455-480. doi: 10.1007/978-1-4939-8958-4_23.
Monoclonal antibodies (mAbs) are currently the fastest growing class of therapeutic proteins. Parallel to full-length IgG format the development of recombinant technologies provided the production of smaller recombinant antibody variants. The single-chain variable fragment (scFv) antibody is a minimal form of functional antibody comprised of the variable domains of immunoglobulin light and heavy chains connected by a flexible linker. In most cases, scFvs are expressed in the periplasm bacterium E. coli. The production of soluble scFvs is more effective in quantity, however, under the reducing conditions of the E. coli bacterial cytoplasm it is inefficient because of the inability of the disulfide bonds to form. Hence, scFvs are either secreted to the periplasm as soluble proteins or expressed in the cytoplasm as insoluble inclusion bodies and recovered by refolding. The cytoplasmic expression of scFvs as a C-terminal fusion to maltose-binding protein (MBP) provided the high-level production of stable, soluble, and functional fusion protein. The below protocol provides the detailed description of MBP-scFv production in E. coli utilizing two expression systems: pMALc-TNN and pMALc-NHNN. Although the MBP tag does not disrupt the most of antibody activities, the MBP-TNN-scFv product can be cleaved by Tobacco Etch Virus (TEV) protease in order to obtain untagged scFv.The second protocol is for efficient production of Fab antibody fragments as MBP fusion proteins secreted by transiently transfected mammalian cells. While transient transfection is a fast and effective way of obtaining several mgs of antibody for initial screening and validation of antibodies, some antibody sequences express poorly or not at all. For such antibodies, fusion to MBP provides an effective approach for solving the expression problem.
单克隆抗体（mAb）是目前治疗性蛋白质中增长最快的类别。与全长IgG形式并行，重组技术的发展使得更小的重组抗体变体得以生产。单链可变片段（scFv）抗体是功能性抗体的最小形式，由免疫球蛋白轻链和重链的可变结构域通过柔性接头连接而成。在大多数情况下，scFv在大肠杆菌周质中表达。可溶性scFv的产量更高，然而，在大肠杆菌细胞质的还原条件下，由于二硫键无法形成，其生产效率低下。因此，scFv要么作为可溶性蛋白质分泌到周质中，要么在细胞质中作为不溶性包涵体表达并通过重折叠回收。将scFv作为麦芽糖结合蛋白（MBP）的C端融合蛋白在细胞质中表达，可实现稳定、可溶性和功能性融合蛋白的高水平生产。以下方案详细描述了利用两种表达系统（pMALc - TNN和pMALc - NHNN）在大肠杆菌中生产MBP - scFv的方法。尽管MBP标签不会破坏大多数抗体活性，但MBP - TNN - scFv产物可通过烟草蚀纹病毒（TEV）蛋白酶切割以获得无标签的scFv。第二个方案是用于高效生产Fab抗体片段，作为瞬时转染哺乳动物细胞分泌的MBP融合蛋白。虽然瞬时转染是获得几毫克抗体用于抗体初步筛选和验证的快速有效方法，但一些抗体序列表达不佳或根本不表达。对于此类抗体，与MBP融合提供了一种解决表达问题的有效方法。
