Disrupting the hydrophobic patches at the antibody variable/constant domain interface: Improved in vivo folding and physical characterization of an engineered scFv fragment
Disrupting the hydrophobic patches at the antibody variable/constant domain interface: Improved in vivo folding and physical characterization of an engineered scFv fragment
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DOI：
10.1093/protein/10.4.435
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发表时间：
1997-04-01
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影响因子：
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通讯作者：
Pluckthun, A
中科院分区：
文献类型：
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作者：
Nieba, L;Honegger, A;Pluckthun, A
通过构建抗体的Fv和单链Fv（scFv）片段，将可变结构域从它们在Fab片段中的天然环境中取出，其中它们与轻链（C-L）和重链（C（H）1）的恒定结构域缔合。因此，前可变/恒定结构域界面的所有残基变得暴露于溶剂。在对30个非冗余Fab结构的分析中，发现在Fv片段的前可变/恒定结构域界面处暴露的疏水残基的频率远高于Fv片段表面的其余部分。我们研究了这些残基对于不同性质的重要性，例如体内和体外折叠、热力学稳定性、天然蛋白的溶解性和抗原亲和力。实验模型系统是抗荧光素抗体4-4-20的scFv片段，当在大肠杆菌的周质中表达时，其中只有2%是天然的。为了改善其在体内的折叠，进行了三个新暴露的界面残基以各种组合的诱变研究，（V-H中的V84 D）导致抗体4-4-20的scFv片段的功能性周质表达产率增加25倍，对于纯化的scFv片段，显示热力学稳定性和抗原结合常数不受这些突变的影响，但是热诱导的聚集反应的速率降低，仅观察到对天然蛋白质的溶解度的微小影响，表明突变防止折叠期间的聚集而不是天然蛋白质的聚集。由于所有scFv片段的构建导致这些残基暴露在先前的可变/恒定结构域界面处，因此该策略应通常适用于改善scFv片段的体内折叠，并且通过类比，也可以在体内折叠其他工程蛋白质结构域。
By constructing Fv and single-chain Fv (scFv) fragments of antibodies, the variable domains are taken out of their natural context in the Fab fragment, where they are associated with the constant domains of the light (C-L) and heavy chain (C(H)1). As a consequence, all residues of the former variable/constant domain interface become solvent exposed. In an analysis of 30 non-redundant Fab structures it was found that at the former variable/constant domain interface of the Fv fragment the frequency of exposed hydrophobic residues is much higher than in the rest of the Fv fragment surface, We investigated the importance of these residues for different properties such as folding in vivo and in vitro, thermodynamic stability, solubility of the native protein and antigen affinity. The experimental model system was the scFv fragment of the anti-fluorescein antibody 4-4-20, of which only 2% is native when expressed in the periplasm of Escherichia coli. To improve its in vivo folding, a mutagenesis study of three newly exposed interfacial residues in various combinations was carried out, The replacement of one of the residues (V84D in V-H) led to a 25-fold increase of the functional periplasmic expression yield of the scFv fragment of the antibody 4-4-20, With the purified scFv fragment it was shown that the thermodynamic stability and the antigen binding constant are not influenced by these mutations, but the rate of the thermally induced aggregation reaction is decreased, Only a minor effect on the solubility of the native protein was observed, demonstrating that the mutations prevent aggregation during folding and not of the native protein, Since the construction of all scFv fragments leads to the exposure of these residues at the former variable/constant domain interface, this strategy should be generally applicable for improving the in vivo folding of scFv fragments and, by analogy, also the in vivo folding of other engineered protein domains.