In vitro sumoylation of recombinant proteins and subsequent purification for use in enzymatic assays.
The sumoylation reaction is mechanistically similar to ubiquitination. It is ATP-dependent and in vitro can be completed in two steps using purified E1 (SAE1/SAE2), E2 (ubc9), and SUMO. Even without the inclusion of an E3 ligase, many substrates can be modified at the same lysines in vitro as in vivo. Here we describe a simplified in vitro sumoylation protocol using recombinant sumoylation substrate, E1, E2, SUMO, and an ATP-regenerating system. The modified substrate can then be repurified from the reaction mixture in a single step to be used in assays to assess the impact of sumoylation on enzymatic and/or other activities.
Vethantham
Vasupradha
V
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Manley
James L
JL
D000255:Adenosine Triphosphate / Q000737:chemistry; D001419:Bacteria / Q000378:metabolism; D005821:Genetic Techniques; D005982:Glutathione Transferase / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D025842:SUMO-1 Protein / Q000737:chemistry*; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism
D011994:Recombinant Proteins; D025842:SUMO-1 Protein; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D044764:Ubiquitin-Activating Enzymes; D008239:Lysine