Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli.
Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli.
复制标题
DOI：
10.1186/s12934-016-0419-5
复制
发表时间：
2016-01-25
影响因子：
6.4
通讯作者：
Ruddock LW
中科院分区：
文献类型：
--
作者：
Gaciarz A;Veijola J;Uchida Y;Saaranen MJ;Wang C;Hörkkö S;Ruddock LW
二硫键是蛋白质中最常见的结构翻译后修饰。根据类型，抗体最多含有 25 个二硫键，其中 scFv 片段含有两个二硫键，Fab 片段含有 5 个或 6 个二硫键。含有天然二硫键的抗体片段的生产可能具有挑战性，尤其是大规模生产。该蛋白质需要靶向原核细胞周质或真核细胞内质网。这些隔室专门用于二硫键的形成，但这两个隔室都有局限性。在这里，我们证明，将二硫键形成催化剂和二硫键异构化催化剂引入细胞质中，可以在大肠杆菌细胞质中有效形成天然折叠的scFv和Fab抗体片段，并具有完整的还原途径。筛选了 11 个 scFv 和 11 个 Fab 片段，并从深孔板中以 > 5 mg/L 的产量获得了各 10 个片段。八个 scFv 和全部十个 Fab 的生产显示出对折叠因子添加的强烈依赖性。纯化的 scFv 产量高达 240 mg/L，纯化的 Fab 片段产量高达 42 mg/L。纯化的片段显示出与天然折叠一致的圆二色光谱并且具有生物活性。我们的结果表明，在大肠杆菌细胞质中有效生产可溶性、具有生物活性的 scFv 和 Fab 抗体片段不仅是可能的，而且是容易的。所需的成分可以在不同的大肠杆菌菌株之间轻松转移。本文的在线版本 (doi:10.1186/s12934-016-0419-5) 包含补充材料，可供授权用户使用。
Disulfide bonds are the most common structural, post-translational modification found in proteins. Antibodies contain up to 25 disulfide bonds depending on type, with scFv fragments containing two disulfides and Fab fragments containing five or six disulfide bonds. The production of antibody fragments that contain native disulfide bonds can be challenging, especially on a large scale. The protein needs to be targeted to prokaryotic periplasm or the eukaryotic endoplasmic reticulum. These compartments are specialised for disulfide bond formation, but both compartments have limitations. Here we show that the introduction into the cytoplasm of a catalyst of disulfide bond formation and a catalyst of disulfide bond isomerization allows the efficient formation of natively folded scFv and Fab antibody fragments in the cytoplasm of Escherichia coli with intact reducing pathways. Eleven scFv and eleven Fab fragments were screened and ten of each were obtained in yields of >5 mg/L from deep-well plates. Production of eight of the scFv and all ten of the Fab showed a strong dependence on the addition of the folding factors. Yields of purified scFv of up to 240 mg/L and yields of purified Fab fragments of up to 42 mg/L were obtained. Purified fragments showed circular dichroism spectra consistent with being natively folded and were biologically active. Our results show that the efficient production of soluble, biologically active scFv and Fab antibody fragments in the cytoplasm of E. coli is not only possible, but facile. The required components can be easily transferred between different E. coli strains. The online version of this article (doi:10.1186/s12934-016-0419-5) contains supplementary material, which is available to authorized users.