Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 511-528 |
| Number of pages | 18 |
| Journal | Methods in Enzymology |
| Volume | 408 |
| DOIs | |
| State | Published - 2006 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
Cite this
RAG and HMGB1 Proteins : Purification and Biochemical Analysis of Recombination Signal Complexes. / Bergeron, Serge; Anderson, Dirk K.; Swanson, Patrick.
In: Methods in Enzymology, Vol. 408, 2006, p. 511-528.Research output: Contribution to journal › Review article
}
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
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
VL - 408
SP - 511
EP - 528
JO - Methods in Enzymology
JF - Methods in Enzymology
SN - 0076-6879
ER -