Qi Peipei, Ding Ying-Ying, He Ting, Yang Tong, Chen Qiuli, Feng Jiaojiao, Wang Jinhong, Cao Mingmei, Li Xiangyu, Peng Heng, Zhu Huaimin, Cao Jie, Pan Wei
Department of Medical Microbiology and Parasitology, School of Basic Medicine, Second Military Medical University, Shanghai 200433, China.
Fudan-Zhangjiang Bio-pharmaceutical Co., Ltd., Shanghai 201203, China.
Sci Rep. 2014 Nov 4;4:6908. doi: 10.1038/srep06908.
Staphylococcus aureus protein A (SpA) and protein G of groups C and G streptococci (SpG) are two well-defined bacterial immunoglobulin (Ig)-binding proteins (IBPs) with high affinity for specific sites on IgG from mammalian hosts. Both SpA and SpG contain several highly-homologous IgG-binding domains, each of which possesses similar binding characteristic of the whole corresponding proteins. Whether specific combinations of these domains could generate a molecule with novel IgG-binding properties remained unknown. We constructed a combinatorial phage library displaying randomly-rearranged A, B, C, D and E domains of SpA as well as the B2 (G2) and B3 (G3) domains of SpG. In vitro molecular evolution directed by human, rabbit, bovine, or goat polyclonal IgGs and four subclasses of mouse monoclonal IgGs generated one common combination, D-C-G3. A series of assays demonstrated that D-C-G3 exhibited a potential novel IgG binding property that was obviously different from those of both parent proteins. This study provides an example of successful protein engineering through in vitro molecular evolution and useful approaches for structure and function studies of IBPs.
金黄色葡萄球菌蛋白A（SpA）以及C组和G组链球菌的蛋白G（SpG）是两种明确的细菌免疫球蛋白（Ig）结合蛋白（IBP），它们对来自哺乳动物宿主的IgG上的特定位点具有高亲和力。SpA和SpG都包含几个高度同源的IgG结合结构域，每个结构域都具有与其相应完整蛋白相似的结合特性。这些结构域的特定组合是否能产生具有新型IgG结合特性的分子仍然未知。我们构建了一个组合噬菌体文库，展示了SpA随机重排的A、B、C、D和E结构域以及SpG的B2（G2）和B3（G3）结构域。由人、兔、牛或山羊多克隆IgG以及小鼠单克隆IgG的四个亚类指导的体外分子进化产生了一种常见组合，即D-C-G3。一系列实验表明，D-C-G3表现出一种潜在的新型IgG结合特性，这与两种亲本蛋白的特性明显不同。本研究提供了一个通过体外分子进化成功进行蛋白质工程的例子，以及用于IBP结构和功能研究的有用方法。
