Preparation of sumoylated substrates for biochemical analysis.
Covalent modification of proteins with SUMO (small ubiquitin related modifier) affects many cellular processes like transcription, nuclear transport, DNA repair and cell cycle progression. Although hundreds of SUMO targets have been identified, for several of them the function remains obscure. In the majority of cases sumoylation is investigated via "loss of modification" analysis by mutating the relevant target lysine. However, in other cases this approach is not successful since mapping of the modification site is problematic or mutation does not cause an obvious phenotype. These latter cases ask for different approaches to investigate the target modification. One possibility is to choose the opposite approach, a "gain in modification" analysis by producing both SUMO modified and unmodified protein in vitro and comparing them in functional assays. Here, we describe the purification of the ubiquitin conjugating enzyme E2-25K, its in vitro sumoylation with recombinant enzymes and the subsequent separation and purification of the modified and the unmodified forms.
Knipscheer
Puck
P
Department of Biological Chemisty and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
Klug
Helene
H
Sixma
Titia K
TK
Pichler
Andrea
A
D001671:Biochemistry / Q000379:methods*; D019411:Clinical Laboratory Techniques; D006801:Humans; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D044763:Ubiquitin-Conjugating Enzymes / Q000302:isolation & purification / Q000378:metabolism*
D011506:Proteins; D011994:Recombinant Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D044763:Ubiquitin-Conjugating Enzymes