Heat-induced Irreversible Denaturation of the Camelid Single Domain VHH Antibody Is Governed by Chemical Modifications
Heat-induced Irreversible Denaturation of the Camelid Single Domain VHH Antibody Is Governed by Chemical Modifications
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DOI：
10.1074/jbc.m113.534222
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发表时间：
2014-05-30
影响因子：
4.8
通讯作者：
Hagihara, Yoshihisa
中科院分区：
文献类型：
--
作者：
Akazawa-Ogawa, Yoko;Takashima, Mizuki;Hagihara, Yoshihisa
骆驼重链抗体的可变区（VHH）具有高度耐热性，因此是许多应用的理想选择。尽管了解热诱导的不可逆变性过程对于提高VHH的功效至关重要，但其失活机制仍不清楚。在这里，我们表明，化学修饰主要控制VHH在高温下的不可逆变性。热处理后，VHH的活性仅取决于90 ℃下的孵育时间，对加热（90 ℃）-冷却（20 ℃）循环的次数不敏感，表明折叠/解折叠中间体对永久变性的作用可以忽略不计。残余活性与浓度无关;因此，VHH以单分子方式而不是通过聚集失去其活性。缺乏Asn的VHH突变体（易受化学修饰的影响）具有比野生型蛋白质显著更高的耐热性，表明化学修饰对VHH变性的重要性。
The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 degrees C and was insensitive to the number of heating (90 degrees C)-cooling (20 degrees C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.