Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5121

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In Vitro Sumoylation of Recombinant Proteins and Subsequent Purification for Use in Enzymatic Assays

Vasupradha Vethantham and James L. Manley1

Department of Biological Sciences, Columbia University, New York, NY 10027, USA

1Corresponding author (jlm2{at}columbia.edu)


INTRODUCTION

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.


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