Mutual stabilization of VL and VH in single-chain antibody fragments, investigated with mutants engineered for stability.
Mutual stabilization of VL and VH in single-chain antibody fragments, investigated with mutants engineered for stability.
复制标题
单链抗体片段中 VL 和 VH 的相互稳定性，通过稳定性工程突变体进行研究。
DOI：
10.1021/bi980712q
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发表时间：
1998
期刊：
影响因子：
2.9
通讯作者：
A. Plückthun
中科院分区：
文献类型：
--
作者：
Arne Wörn;A. Plückthun
产生了一组六个莱万结合单链Fv（scFv）片段A48（ABPC 48）的突变体，其具有相同的轻链，但重链的稳定性逐渐不同。这通过将稳定化突变H-K66 R和H-N52 S中的一个或两个引入A48野生型蛋白的VH结构域（其天然缺失VH中的保守二硫键）和半胱氨酸恢复的变体A48 cys scFv中来实现。这两个突变在VH中的稳定作用，其已经在该scFv片段的无二硫化物衍生物的背景下选择[Proba，K.，等人（1998）J. Mol. Biol.275，245-253]，被发现对A48 scFv的半胱氨酸恢复变体是加和的并且可转移的，从而产生极其稳定的VH结构域。将这些scFv片段的平衡变性与相应的分离的VL结构域和两个不同的分离的VH结构域进行比较。在scFv片段中，发现VL结构域被更稳定的VH结构域稳定，并且相反地，VH结构域被更稳定的VL结构域稳定。一个折叠中间体与天然样VH和变性VL被发现在平衡，如果VH是显着更稳定比VL。在所有其他情况下，观察到scFv的协同解折叠。我们用单链抗体中的伴侣结构域提供的内在结构域稳定性和外在稳定性的不同贡献来解释这一观察结果。
A set of six mutants of the levan binding single-chain Fv (scFv) fragment A48 (ABPC48), which have the identical light chain but differ gradually in the stability of the heavy chain, was generated. This was achieved by introducing one or both of the stabilizing mutations H-K66R and H-N52S into the VH domain of the A48 wild-type protein, which is naturally missing the conserved disulfide bridge in VH, and into the cysteine-restored variant A48cys scFv. The stabilizing effects of these two mutations in VH, which had been selected in the context of a disulfide-free derivative of this scFv fragment [Proba, K., et al. (1998) J. Mol. Biol. 275, 245-253], were found to be additive and transferable to the cysteine-restored variant of the A48 scFv, thereby generating extremely stable VH domains. The equilibrium denaturation of these scFv fragments was compared with the corresponding isolated VL domain and two of the different isolated VH domains. In the scFv fragment, the VL domain was found to be stabilized by a more stable VH domain, and, conversely, the VH domain was stabilized by a more stable VL domain. A folding intermediate with nativelike VH and denatured VL was found at equilibrium, if VH was significantly more stable than VL. In all other cases, a cooperative unfolding of the scFv was observed. We explain this observation with different contributions of intrinsic domain stability and extrinsic stabilization provided by the partner domain in the single-chain antibodies.