Thermodynamically stable aggregation-resistant antibody domains through directed evolution
Thermodynamically stable aggregation-resistant antibody domains through directed evolution
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DOI：
10.1016/j.jmb.2007.10.075
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发表时间：
2008-02-29
影响因子：
5.6
通讯作者：
Winter, Greg
中科院分区：
文献类型：
--
作者：
Famm, Kristoffer;Hansen, Lars;Winter, Greg
蛋白质聚集体通常由未折叠或部分未折叠的物种之间的相互作用形成，并且通常发生在蛋白质变性时，例如，热或低pH。在早期的工作中，我们使用达尔文选择策略来创建抵抗热聚集的人抗体可变结构域。将结构域库展示在丝状噬菌体上并变性（在80 ℃，pH 7.4），冷却后通过与通用配体结合来选择折叠结构域。该过程似乎选择了具有抵抗聚集的变性状态的结构域，但结构域仅具有低折叠自由能（在25 ℃，pH 7.4下，Δ G度（N-D）= 15-20 kJ/mol）。在这里，使用相同的噬菌体库，我们已经将该方法扩展到抗酸聚集的结构域的选择。然而，在这种情况下，所选择的结构域的热力学稳定性高于通过热变性所选择的结构域（在中性和酸性条件下;在pH 7.4下在25 ℃下Δ G度（N-D）=26-47 kJ/mol，或在pH 3.2下Δ G度（N-D）= 27-34 kJ/mol）。此外，我们确定了一个关键的决定因素（Arg 28），增加了聚集阻力的变性状态的域在低pH值，而不损害其热力学稳定性。因此，选择过程产生了结合热力学稳定性和抗聚集未折叠状态的结构域。我们认为，这些属性的变化是由折叠平衡在选择过程中被取代的程度控制的。(c)2007爱思唯尔有限公司保留所有权利。
Protein aggregates are usually formed by interactions between unfolded or partially unfolded species, and often occur when a protein is denatured by, for example, heat or low pH. In earlier work, we used a Darwinian selection strategy to create human antibody variable domains that resisted heat aggregation. The repertoires of domains were displayed on fila mentous phage and denatured (at 80 degrees C in pH 7.4), and folded domains were selected by binding to a generic ligand after cooling. This process appeared to select for domains with denatured states that resisted aggregation, but the domains only had low free energies of folding (Delta G degrees(N-D) = 15-20 kJ/mol at 25 degrees C in pH 7.4). Here, using the same phage repertoire, we have extended the method to the selection of domains resistant to acid aggregation. In this case, however, the thermodynamic stabilities of selected domains were higher than those selected by thermal denaturation (under both neutral and acidic conditions; Delta G degrees(N-D)=26-47 kJ/mol at 25 degrees C in pH 7.4, or Delta G degrees(N-D) = 27-34 kJ/mol in pH 3.2). Furthermore, we identified a key determinant (Arg28) that increased the aggregation resistance of the denatured states of the domains at low pH without compromising their thermodynamic stabilities. Thus, the selection process yielded domains that combined thermodynamic stability and aggregation-resistant unfolded states. We suggest that changes to these properties are controlled by the extent to which the folding equilibrium is displaced during the process of selection. (c) 2007 Elsevier Ltd. All rights reserved.