Expression of an antibody fragment at high levels in the bacterial cytoplasm
Expression of an antibody fragment at high levels in the bacterial cytoplasm
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在细菌细胞质中高水平表达抗体片段
DOI：
10.1006/jmbi.1998.1840
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发表时间：
1998-07-03
影响因子：
5.6
通讯作者：
Winter, G
中科院分区：
文献类型：
--
作者：
Martineau, P;Jones, P;Winter, G
在细胞质中表达的重组抗体片段具有相当大的实用潜力。然而，在细胞质的还原环境中，不形成结构域内二硫键，并且片段不稳定并且以低产量表达。我们试图克服这些局限性。我们首先分离了结合并激活失活突变体β-半乳糖苷酶的抗体单链Fv片段。然后，我们在体外通过易错聚合酶链反应对编码scFv片段的基因进行随机突变，并在lac（-）细菌中共表达突变的β-半乳糖苷酶和突变的抗体片段。通过在限制性乳糖上铺板，我们选择了具有改善的表达的抗体突变体，并且在连续四轮突变和选择之后，分离了在细菌细胞质中表达的抗体片段，其在摇瓶中的产率为0.5g/l（A（600 nm）为5.5），在发酵罐中的产率为3.1g/l（A（600 nm）= 33）。对突变抗体片段的分析表明，二硫键在细胞质中被还原，并且片段可以在体外还原条件下有效地变性和复性。这表明，使用合适的筛选或选择方法，可以在细胞质中以优异的产率制备折叠的和功能性的抗体片段。(C)北京：科学出版社.
Recombinant antibody fragments expressed in the cytoplasm of cells have considerable practical potential. However in the reducing environment of the cytoplasm, the intradomain disulphide bonds are not formed and the fragments are unstable and expressed in low yields. Here we attempted to overcome these limitations. We first isolated an antibody single chain Fv fragment that binds and activates an inactive mutant beta-galactosidase. We then subjected the gene encoding the scFv fragment to random mutation in vitro by error-prone polymerase chain reaction, and co-expressed the mutant beta-galactosidase and mutant antibody fragments in lac(-) bacteria. By plating on limiting lactose, we selected for antibody mutants with improved expression, and after four successive rounds of mutation and selection, isolated an antibody fragment that is expressed in the bacterial cytoplasm with yields of 0.5 g/l in a shaker flask (A(600 nm) of 5.5) and 3.1 g/l (A(600 nm) = 33) in a fermentor. Analysis of the mutant antibody fragments revealed that the disulphide bonds are reduced in the cytoplasm, and that the fragments could be denatured and renatured efficiently under reducing conditions in vitro. This shows that with a suitable method of screening or selection, it is possible to make folded and functional antibody fragments in excellent yield in the cytoplasm. (C) 1998 Academic Press.