Bergeron Serge, Anderson Dirk K, Swanson Patrick C
Creighton University, Medical Microbiology and Immunology, Omaha, Nebraska, USA.
Methods Enzymol. 2006;408:511-28. doi: 10.1016/S0076-6879(06)08032-3.
Two lymphoid cell-specific proteins, called RAG-1 and RAG-2, initiate the process of antigen receptor gene rearrangement, termed V(D)J recombination, by assembling a protein-DNA complex with two recombination signal sequences (RSSs), each of which adjoins a different receptor gene segment, and then introducing a DNA double strand break at the end of each RSS. The study of RAG-RSS complex assembly and activity has been facilitated by the development of methods to purify the RAG proteins and members of the HMG-box family of high mobility group proteins such as HMGB1 that promote RAG binding and cleavage activity in vitro. This chapter describes the purification of recombinant truncated and full-length RAG-1 and RAG-2 expressed transiently in mammalian cells, as well as the purification of bacterially expressed full-length HMGB1. In addition, it details several experimental procedures used in our laboratory to study RAG-RSS complex formation and function in vitro.
两种淋巴细胞特异性蛋白，即RAG-1和RAG-2，通过与两个重组信号序列（RSS）组装成蛋白质-DNA复合物来启动抗原受体基因重排过程，即V(D)J重组，每个重组信号序列毗邻一个不同的受体基因片段，然后在每个RSS末端引入DNA双链断裂。RAG蛋白和高迁移率族蛋白HMG-box家族成员（如HMGB1）的纯化方法的发展促进了对RAG-RSS复合物组装和活性的研究，这些蛋白在体外可促进RAG的结合和切割活性。本章描述了在哺乳动物细胞中瞬时表达的重组截短型和全长RAG-1和RAG-2的纯化，以及细菌表达的全长HMGB1的纯化。此外，还详细介绍了我们实验室用于研究体外RAG-RSS复合物形成和功能的几个实验程序。
