RAG and HMGB1 proteins: purification and biochemical analysis of recombination signal complexes

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.