Xu Qin, Xie Zhiqun, Ding Jianfang, Lin Sheng-Xiang, Xu Genjun
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China.
Protein Sci. 2004 Jul;13(7):1851-8. doi: 10.1110/ps.04699904.
The reactivation efficiency in the refolding of denatured luciferase in the presence and the absence of monoclonal antibodies (mAbs) has been studied. Luciferase could be partially reactivated when the protein was denatured in high concentrations of guanidium chloride (GdmCl; >4.5 M) and the refolding was carried out in very low protein concentrations. The refolding yield was, however, significantly lower when it was performed on luciferase that had been denatured with lower concentrations of GdmCl. The efficiency of refolding decreases when the formation of aggregates increases. Three of the five luciferase mAbs tested (4G3, N2E3, S2G10) dramatically increased the yield of reactivation and simultaneously eliminated the formation of aggregates. It is proposed that these mAbs assisted the refolding of luciferase by binding to the exposed hydrophobic surface of the refolding intermediate, thus preventing it from aggregating. The epitopes interacting with these refolding-assisting mAbs are all located in the A-subdomain of the N-terminal region of luciferase. These results have also shed light on the structural features of the intermediate and its interface involved in protein aggregate formation, contributing to the understanding of the protein folding mechanism.
研究了在有和没有单克隆抗体（mAb）存在的情况下，变性荧光素酶复性中的再激活效率。当蛋白质在高浓度胍盐酸盐（GdmCl；>4.5 M）中变性，且在非常低的蛋白质浓度下进行复性时，荧光素酶可以部分再激活。然而，当对用较低浓度GdmCl变性的荧光素酶进行复性时，复性产率显著降低。当聚集体形成增加时，复性效率降低。所测试的五种荧光素酶单克隆抗体中的三种（4G3、N2E3、S2G10）显著提高了再激活产率，同时消除了聚集体的形成。有人提出，这些单克隆抗体通过与复性中间体暴露的疏水表面结合来协助荧光素酶复性，从而防止其聚集。与这些协助复性的单克隆抗体相互作用的表位都位于荧光素酶N端区域的A亚结构域中。这些结果也揭示了参与蛋白质聚集体形成的中间体及其界面的结构特征，有助于理解蛋白质折叠机制。
