Maeda Y, Ueda H, Kazami J, Kawano G, Suzuki E, Nagamune T
Department of Chemistry and Biotechnology, Faculty of Engineering, University of Tokyo, Japan.
Anal Biochem. 1997 Jul 1;249(2):147-52. doi: 10.1006/abio.1997.2181.
Luciferase of Vargula hilgendorfli is infinitely stable at room temperature in dried state, and its light-emitting reaction is very simple. These unique characteristics of Vargula luciferase have prompted us to engineer chimeric protein, the other moiety chosen for conjugation being streptococcal protein G. A single domain of protein G which binds to IgG of a wide range of species was fused at the N-terminal region of Vargula luciferase. Unexpectedly, we found that the chimeric protein expressed in mammalian COS-1 cells had no IgG-binding ability, probably due to some sort of interaction between the two moieties or some conformational preferences of the IgG-binding domain of protein G when fused to Vargula luciferase. Here we report how we regained the IgG binding of protein G, by the intervention of three alpha-helices of protein A between protein G and luciferase. To our knowledge, the new chimeric protein provides the first reported model of this kind.
希氏软腹栉水母的荧光素酶在干燥状态下于室温下具有无限稳定性，并且其发光反应非常简单。希氏软腹栉水母荧光素酶的这些独特特性促使我们构建嵌合蛋白，选择与之结合的另一部分是链球菌蛋白G。蛋白G的一个与多种物种的IgG结合的单一结构域在希氏软腹栉水母荧光素酶的N端区域融合。出乎意料的是，我们发现，在哺乳动物COS-1细胞中表达的嵌合蛋白没有IgG结合能力，这可能是由于两个部分之间的某种相互作用，或者是蛋白G的IgG结合结构域与希氏软腹栉水母荧光素酶融合时的某种构象偏好。在此我们报告，通过在蛋白G和荧光素酶之间插入蛋白A的三个α螺旋，我们如何恢复了蛋白G的IgG结合能力。据我们所知，这种新的嵌合蛋白提供了首个此类报道模型。
