| Chapter title |
Reconstitution of the Recombinant RanBP2 SUMO E3 Ligase Complex.
|
|---|---|
| Chapter number | 3 |
| Book title |
SUMO
|
| Published in |
Methods in molecular biology, January 2016
|
| DOI | 10.1007/978-1-4939-6358-4_3 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-6356-0, 978-1-4939-6358-4
|
| Authors |
Tobias Ritterhoff, Hrishikesh Das, Yuqing Hao, Volkan Sakin, Annette Flotho, Andreas Werner, Frauke Melchior |
| Editors |
Manuel S. Rodriguez |
| Abstract |
One of the few proteins that have SUMO E3 ligase activity is the 358 kDa nucleoporin RanBP2 (Nup358). While small fragments of RanBP2 can stimulate SUMOylation in vitro, the physiologically relevant E3 ligase is a stable multi-subunit complex comprised of RanBP2, SUMOylated RanGAP1, and Ubc9. Here, we provide a detailed protocol to in vitro reconstitute the RanBP2 SUMO E3 ligase complex. With the exception of RanBP2, reconstitution involves untagged full-length proteins. We describe the bacterial expression and purification of all complex components, namely an 86 kDa His-tagged RanBP2 fragment, the SUMO E2-conjugating enzyme Ubc9, RanGAP1, and SUMO1, and we provide a protocol for quantitative SUMOylation of RanGAP1. Finally, we present details for the assembly and final purification of the catalytically active RanBP2/RanGAP1*SUMO1/Ubc9 complex. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 11 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 3 | 27% |
| Student > Ph. D. Student | 2 | 18% |
| Unspecified | 1 | 9% |
| Unknown | 5 | 45% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 5 | 45% |
| Unspecified | 1 | 9% |
| Unknown | 5 | 45% |