Different equilibrium stability behavior of ScFv fragments:: Identification, classification, and improvement by protein engineering
Different equilibrium stability behavior of ScFv fragments:: Identification, classification, and improvement by protein engineering
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DOI：
10.1021/bi9902079
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发表时间：
1999-07-06
期刊：
影响因子：
2.9
通讯作者：
Plückthun, A
中科院分区：
文献类型：
--
作者：
Wörn, A;Plückthun, A
提出了关于 scFv 片段的解折叠/重折叠平衡的分类。它基于对果聚糖结合 A48 scFv 片段和 HER-2 结合 4D5 scFv 片段的不同突变体以及携带 4D5 V-L 结构域和 A48 突变体 V-H 结构域的“混合”scFv 的分析。测量相应 scFv 片段的变性剂诱导的去折叠曲线，如果需要分类，则与分离结构域的变性进行比较。根据结构域的相对内在稳定性和界面的稳定性，不同的 scFv 片段被分为不同的类别。我们还通过几个例子证明了如何通过将工程工作集中在特定分子的“薄弱部分”（可能是 V-L、V-H 的内在稳定性或界面的稳定性），使用这种分类来提高给定 scFv 片段的稳定性。通过这种方法获得的 scFv 片段之一非常稳定，仅在约 7 M 尿素时开始变性。我们相信这种极其稳定的框架可能是CDR移植实验中非常合适的受体。此外，七个相关 A48 scFv 突变体的热力学平衡稳定性涵盖了尿素解折叠的广泛稳定性，与通过光散射和分析凝胶过滤测量的热聚集特性密切相关。
A classification of scFv fragments concerning their unfolding/refolding equilibria is proposed. It is based on the analysis of different mutants of the levan-binding A48 scFv fragment and the HER-2 binding 4D5 scFv fragment as well as a "hybrid" scFv carrying the V-L domain of 4D5 and the V-H domain of an A48 mutant. The denaturant-induced unfolding curves of the corresponding scFv fragments were measured and, if necessary for the classification, compared with the denaturation of the isolated domains. Depending on the relative intrinsic stabilities of the domains and the stability of the interface, the different scFv fragments were grouped into different classes. We also demonstrate with several examples how such a classification can be used to improve the stability of a given scFv fragment, by concentrating engineering efforts on the "weak part" of the particular molecule, which may either be the intrinsic stability of V-L, Of V-H, or the stability of the interface. One of the scFv fragments obtained by this kind of approach is extremely stable, starting denaturation only at about 7 M urea. We believe that such extremely stable frameworks may be very suitable recipients in CDR grafting experiments. In addition, the thermodynamic equilibrium stabilities of seven related A48 scFv mutants covering a broad range of stabilities in urea unfolding were shown to be well correlated with thermal aggregation properties measured by light scattering and analytical gel filtration.