Abstract
Posttranslational modification by small ubiquitin-like modifiers (SUMO) is being associated with a growing number of regulatory functions in diverse cellular processes. The biochemical investigation into the underlying molecular mechanisms, however, has been lagging behind due to the difficulty to generate sufficient amounts of recombinant SUMOylated proteins. Here, we present two newly designed two-component vector systems for the expression and purification of SUMO-modified target proteins in Escherichia coli. One system consists of a vector for SUMO conjugation, expressing human SUMO-activating (SAE1/SAE2) and conjugating (Ubc9) enzymes together with His6-tagged SUMO1, 2 or 3, that can be combined with commonly used expression constructs for any gene of interest. To facilitate SUMOylation of targets normally requiring a SUMO-E3 ligase for efficient modification, a second system is designed to express the target protein as a fusion with the human SUMO-conjugating enzyme Ubc9, thus compensating the absence of a potential SUMO ligase. We demonstrate the proficiency of these systems by SUMOylation of two DNA repair proteins, the thymine DNA glycosylase (TDG) and XRCC1, and describe purification schemes for SUMOylated proteins in native and active form. This SUMO toolbox facilitates “in-cell” and “in-extract” production and purification of recombinant SUMO-modified target proteins for functional and structural analysis.
Highlights
Posttranslational modification by ubiquitin-like polypeptides, socalled UBLs, affects a large number of proteins, thereby regulating a variety of cellular processes [1,2]
Unique to this system is that it bases on human proteins only, expresses the heterodimeric SAE1-SAE2 complex (SUMO-E1) from a bicistronic unit and provides small ubiquitin-like modifiers (SUMO) polypeptides with an Nterminal His6-tag separated by a thrombin cleavage site, facilitating the enrichment of modified proteins by affinity chromatography and the removal of the affinity tag
To ensure a stable maintenance of the pSUMO1 and the target expression constructs in E.coli, we chose pMB1ori-based plasmids for the expression of the mouse thymine DNA glycosylase (TDG) and the human XRCC1 (Figure 1C)
Summary
Posttranslational modification by ubiquitin-like polypeptides, socalled UBLs, affects a large number of proteins, thereby regulating a variety of cellular processes [1,2]. The SUMO (small ubiquitinlike modifier) peptides represent a prominent subfamily of the UBLs and exist in four different isoforms (SUMO1, SUMO2, SUMO3 and SUMO4) in mammalian cells, each encoded by a different gene. These SUMOs differ to some extent in their amino acid sequences – SUMO2 and SUMO3 share sequence identity of 97% with each other and about 50% with SUMO1 – they all show high 3D-structural resemblance [3,4,5,6]. SUMO conjugation is promoted by SUMO-E3 ligases, which act as substrate-specific adapters (Figure 1A)
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