Effect of an intermolecular disulfide bond introduced into the first loop of CH1 domain of Adalimumab Fab on thermal stability and antigen-binding activity
Effect of an intermolecular disulfide bond introduced into the first loop of CH1 domain of Adalimumab Fab on thermal stability and antigen-binding activity
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阿达木单抗Fab CH1结构域第一环引入分子间二硫键对热稳定性和抗原结合活性的影响
DOI：
10.1093/jb/mvac040
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Ohkuri T
中科院分区：
文献类型：
--
作者：
Yoshikawa M;Nakamura H;Oda-Ueda N;Ueda T;Ohkuri T
氨基酸突变引入分子间二硫键是稳定二聚体蛋白的有效方法。治疗性抗体阿达木单抗Fab的x射线晶体结构显示，CH1结构域的第一个环在135-141位部分未解。为了在阿达木单抗Fab中找到引入分子间二硫键的新位点，利用分子模拟软件预测了靶向未解决区域的Fab突变体。4个Fab突变体H:K137C-L:I117C、H:K137C-L:F209C、H:S138C-L:F116C和H:S140C-L:S114C在甲基营养酵母毕赤酵母中表达。SDS-PAGE分析表明，H:K137C-L:F209C、H:S138C-L:F116C和H:S140C-L:S114C突变体主要形成分子间二硫键，而H:K137C-L:I117C突变体部分形成分子间二硫键，部分不形成。差示扫描量热法测量显示，所有具有工程二硫键的Fab突变体的热稳定性都有所提高。结合抗原肿瘤坏死因子α的生物层干涉测量表明，Fab突变体的抗原结合活性低于野生型Fab。其中，H:K137C-L:F209C的kd值比野生型Fab高出约17倍。因此，我们成功地在CH1和CL结构域的第一个环区之间引入了分子间二硫键，并观察到它增加了Fab的热稳定性并影响了抗原结合活性。
The introduction of intermolecular disulfide bonds by amino acid mutations is an effective method for stabilizing dimeric proteins. X-ray crystal structure of Fab of a therapeutic antibody, adalimumab, revealed the first loop of the CH1 domain to be partially unsolved at position 135–141. To find new sites for the introduction of intermolecular disulfide bonds in adalimumab Fab, Fab mutants targeting the unsolved region were predicted using molecular simulation software. Four Fab mutants, H:K137C-L:I117C, H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C, were expressed in the methylotrophic yeastPichia pastoris.SDS-PAGE analysis of these mutants indicated that H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C mutants mostly formed intermolecular disulfide bonds, whereas some H:K137C-L:I117C mutants formed intermolecular disulfide bonds and some did not. Differential scanning calorimetry measurements showed increased thermal stability in all Fab mutants with engineered disulfide bonds. The bio-layer interferometry measurements, for binding of the antigen tumor necrotic factor α, indicated that Fab mutants had less antigen-binding activity than wild-type Fab. In particular, theKDvalue of H:K137C-L:F209C was ~17 times higher than that of wild-type Fab. Thus, we successfully introduced intermolecular disulfide bonds between the first loop region of the CH1 and CL domains and observed that it increases the thermostability of Fab and affects the antigen-binding activity.