Production of Stabilized Antibody Fragments in the E. coli Bacterial Cytoplasm and in Transiently Transfected Mammalian Cells
Production of Stabilized Antibody Fragments in the E. coli Bacterial Cytoplasm and in Transiently Transfected Mammalian Cells
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DOI：
10.1007/978-1-4939-8958-4_23
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发表时间：
2019-01-01
期刊：
影响因子：
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通讯作者：
Benhar, Itai
中科院分区：
文献类型：
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作者：
Birnboim-Perach, Racheli;Grinberg, Yehudit;Benhar, Itai
单克隆抗体（mAb）是目前增长最快的一类治疗性蛋白质。与全长IgG形式平行，重组技术的发展提供了更小的重组抗体变体的生产。单链可变片段（scFv）抗体是由通过柔性接头连接的免疫球蛋白轻链和重链的可变结构域组成的功能性抗体的最小形式。在大多数情况下，scFv在周质细菌E.杆菌可溶性scFv的产生在数量上更有效，然而，在E.由于不能形成二硫键，因此在大肠杆菌细胞质中是低效的。因此，scFv或者作为可溶性蛋白分泌到周质中，或者作为不溶性包涵体在细胞质中表达并通过重折叠回收。作为麦芽糖结合蛋白（MBP）的C-末端融合物的scFv的胞质表达提供了稳定的、可溶的和功能性融合蛋白的高水平生产。以下方案提供了在E.利用pMALc-TNN和pMALc-NHNN两种表达系统在大肠杆菌中进行表达。虽然MBP标签不会破坏抗体的大部分活性，但是MBP-TNN-scFv产物可以被烟草蚀纹病毒（TEV）蛋白酶切割以获得未标记的scFv。第二个方案是用于由瞬时转染的哺乳动物细胞分泌的作为MBP融合蛋白的Fab抗体片段的有效生产。虽然瞬时转染是获得数mg抗体用于抗体的初始筛选和验证的快速且有效的方式，但一些抗体序列表达差或根本不表达。对于这样的抗体，与MBP的融合提供了解决表达问题的有效方法。
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.