Jendeberg L, Nilsson P, Larsson A, Denker P, Uhlén M, Nilsson B, Nygren P A
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
J Immunol Methods. 1997 Feb 14;201(1):25-34. doi: 10.1016/s0022-1759(96)00215-3.
A system for production of recombinant Fc fragments of human IgG in Escherichia coli has been developed to allow for structural and functional studies of human Fc. The genes for the Fc fragments of human IgG subclasses 1 and 3, designated Fc(1) and Fc(3), were cloned from a human spleen cDNA library. The interactions to Staphylococcal protein A (SpA), a bacterial Fc receptor, that interacts with human IgG-Fc(1), but not with human IgG-Fc(3), were analyzed. To corroborate the involvement of amino acid residues in Fc, responsible for these differences in binding, two Fc variants were constructed; Fc(1(3)) and Fc(3(1)), each containing an isotypic dipeptide substitution. Production levels in E. coli of 1-10 mg/l of secreted Fc proteins, covalently linked as dimers, were routinely obtained. SpA-binding analyses of all four Fc variants using biosensor technology, showed that Fc(1) and Fc(3(1)) interact with SpA, while Fc(3) and Fc(1(3)) lack detectable SpA binding. The rendered SpA binding of the Fc variant Fc(3(1)), is concluded to result from the introduced dipeptide substitution (R435H, F436Y). The results demonstrate that the Fc expression system efficiently can be used in Fc engineering.
已开发出一种在大肠杆菌中生产人IgG重组Fc片段的系统，以用于人Fc的结构和功能研究。从人脾脏cDNA文库中克隆了人IgG亚类1和3的Fc片段基因，分别命名为Fc(1)和Fc(3)。分析了与细菌Fc受体葡萄球菌蛋白A（SpA）的相互作用，SpA与人IgG-Fc(1)相互作用，但不与人IgG-Fc(3)相互作用。为了证实Fc中负责这些结合差异的氨基酸残基的作用，构建了两个Fc变体；Fc(1(3))和Fc(3(1))，每个都含有一个同型二肽取代。在大肠杆菌中，通常可获得1-10mg/l以二聚体形式共价连接的分泌型Fc蛋白的产量。使用生物传感器技术对所有四种Fc变体进行的SpA结合分析表明，Fc(1)和Fc(3(1))与SpA相互作用，而Fc(3)和Fc(1(3))缺乏可检测到的SpA结合。得出结论，Fc变体Fc(3(1))呈现的SpA结合是由引入的二肽取代（R435H，F436Y）导致的。结果表明，Fc表达系统可有效地用于Fc工程。
