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.
Bergeron
Serge
S
Creighton University, Medical Microbiology and Immunology, Omaha, Nebraska, USA.
Anderson
Dirk K
DK
Swanson
Patrick C
PC
D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D024243:HMGB1 Protein / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D051379:Mice; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic
D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D018398:Homeodomain Proteins; D021382:Protein Sorting Signals; C499308:Rag2 protein, mouse; D011994:Recombinant Proteins; C064658:RAG-1 protein