TY - JOUR
T1 - RAG and HMGB1 Proteins
T2 - Purification and Biochemical Analysis of Recombination Signal Complexes
AU - Bergeron, Serge
AU - Anderson, Dirk K.
AU - Swanson, Patrick C.
N1 - Funding Information:
This work has been supported by grants to P.C.S. from the American Cancer Society (RSG‐01‐020‐01‐CCE) and the National Institutes of Health (R01 AI055599). Laboratory renovation was funded by the Research Facilities Improvement Program of the NIH National Center for Research Resources (C06 RR17417‐01).
PY - 2006
Y1 - 2006
N2 - 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.
AB - 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.
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U2 - 10.1016/S0076-6879(06)08032-3
DO - 10.1016/S0076-6879(06)08032-3
M3 - Review article
C2 - 16793390
AN - SCOPUS:33745186676
VL - 408
SP - 511
EP - 528
JO - Methods in Enzymology
JF - Methods in Enzymology
SN - 0076-6879
ER -