Nakamura Hitomi, Oda-Ueda Naoko, Ueda Tadashi, Ohkuri Takatoshi
Faculty of Pharmaceutical Sciences, Sojo University, Japan.
Graduate School of Pharmaceutical Sciences, Kyushu University, Japan.
Biochem Biophys Res Commun. 2018 Jan 1;495(1):7-11. doi: 10.1016/j.bbrc.2017.10.140. Epub 2017 Oct 31.
We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH and CL domains was deleted by substitution of Cys with Ala (Fab). DSC measurements showed that the Tm values of Fab were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab. The resulting Fab (mutSS Fab) had the mutations H:V177C and L:Q160C in Fab, confirming the formation of the disulfide bond between CH and CL. The thermostability of mutSS Fab was approximately 5 °C higher than that of Fab. Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation.
我们构建了一个系统，用于在甲基营养型酵母毕赤酵母中以20 mg/L的产量表达治疗性人单克隆抗体阿达木单抗的Fab片段。为了研究链间二硫键对构象稳定性的贡献，我们制备了阿达木单抗Fab片段，通过将半胱氨酸替换为丙氨酸，删除了CH和CL结构域C末端区域的链间二硫键（Fab）。差示扫描量热法（DSC）测量表明，Fab的熔点（Tm）值比野生型Fab低约5°C，这表明链间二硫键有助于构象热稳定性。通过计算机模拟，我们在C末端区域之外设计了一种新型链间二硫键，以提高Fab的稳定性。所得的Fab（mutSS Fab）在Fab中有H:V177C和L:Q160C突变，证实了CH和CL之间二硫键的形成。mutSS Fab的热稳定性比Fab高约5°C。因此，引入设计的链间二硫键增强了Fab的热稳定性，并减轻了C末端区域链间二硫键部分还原所导致的去稳定化，这种去稳定化发生在诸如聚乙二醇化等位点特异性修饰中。
