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SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.
Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.
The purpose of this method is to facilitate the study and analysis of SUMO-conjugated proteins using the recombinant SUMO-trapping UTAG (Ulp domain Tag) protein. As detailed below, UTAG can be used in lieu of other reagents and approaches to purify, detect, and visualize SUMO-modified proteins. Depending on growth conditions, cells may contain hundreds or thousands of proteins that are modified with SUMO or SUMO chains (for review see Kerscher et al. 20061 and Kerscher 20162). This represents a considerable difficulty for the functional analyses of specific SUMO-modified proteins, especi....
1. SUMO detection in fixed tissue culture cells using recombinant KmUTAG-FL SUMO-trapping protein
KmUTAG-fl is a recombinant, mCherry-tagged SUMO-trapping protein. To produce kmUTAG-fl, we cloned a codon-optimized mCherry-kmUTAG into the pSPOT1 bacterial overexpression plasmid (Figure 1). After induction, the kmUTAG-fl protein was purified on Spot-TRAP, eluted, and frozen until further use. To ensure the SUMO-trapping activity of KmUTAG-fl, we confirmed binding to SUMO1-conjugated beads and precipitation of a SUMO-CAT fusion protein (data not shown, but s.......
Here we introduce the use of kmUTAG-fl, a recombinant protein, for functional studies of SUMO in fixed mammalian cells and dissected nematode gonads. KmUTAG-fl is a stress-tolerant pan-SUMO specific reagent that recognizes and traps native SUMO-conjugated proteins and SUMO chains. Since SUMO's tertiary structure is highly conserved it is very likely that SUMO variants from additional model and non-model systems can be analyzed with the kmUTAG-fl reagent. As such, KmUTAG-fl may represent a useful alternative or second.......
We would like to thank all members of the Kerscher lab for their support, Nathalie Nguyen for critical reading of the manuscript, and Lidia Epp for sequencing. This work has been supported by the Commonwealth Research Commercialization fund MF16-034-LS to OK. Research support for W&M students was provided by the Bailey-Huston Research fund, and Charles Center Honors Fellowships to RY and CH.
....Name | Company | Catalog Number | Comments |
16% Paraformaldehyde (formaldehyde) aqueous solution | Electron Microscopy Sciences | 30525-89-4 | |
6-Well Cell Culture Plates | Genesee Scientific/Olympus Plastics | 25-105 | |
Alexa Fluor 488 AffiniPure Goat Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 115-545-003 | Used as a secondary antibody for mouse monoclonal antibody |
DPBS, no calcium, no magnesium | Fisher Scientific | Gibco 14190144 | |
Dylight 488 conjugated AffiniPure Goat Anti-Moue IgG (H+L) | Jackson ImmunoResearch | 115-485-146 | Used as a secondary antibody for mouse monoclonal antibody |
Fisherbrand Coverglass for Growth Cover Glasses | Fisherbrand | 12545101 | |
FLUORO-GEL II with DAPI | Electron Microscopy Sciences | 50-246-93) | Mounting media in step 1.11 |
FLUORO-GEL with DABCO | Electron Microscopy Sciences | 17985-02 | With DAPI added to 1 µg/mL; mounting media in step 2.2.5 |
Glycine-HCl | Fisher BioReagents | BP3815 | |
Glycine-HCl | ACROS Organics | 6000-43-7 | |
KmUTAG-fl | Kerafast | KmUTAG reagents are available on Kerafast.com | |
Oneblock Western-CL blocking buffer | Prometheus | 20-313 | |
PBS, Phosphate Buffered Saline, 10X Solution | Fisher BioReagents | BP3994 | |
PNT2 cell line | Sigma-Aldrich | 95012613 | Normal prostate epithelium immortalized with SV40. |
pSPOT1 | (ChromoTek GmbH) | ev-1 | https://www.chromotek.com/fileadmin/user_upload/pdfs/Datasheets/pSpot1_v1.pdf |
SUMO 6F2 | DSHB | SUMO 6F2 | SUMO 6F2 was deposited to the DSHB by Pelisch, F. / Hay, R.T. (DSHB Hybridoma Product SUMO 6F2) |
SUMO protease buffer [10x] | 500 mM Tris-HCl, pH 8.0, 2% NP-40, 1.5 M NaCl | ||
SUMO-2 Antibody 8A2 | DSHB | SUMO-2 8A2 | SUMO-2 8A2 was deposited to the DSHB by Matunis, M. (DSHB Hybridoma Product SUMO-2 8A2) |
TCEP-HCL | GoldBio | 51805-45-9 | Used as a reducing agent at a concentration of 5mM |
Triton X-100 | Fisher BioReagents | 9002-93-1 | Used for permeablization at 0.1% in DPBS/PBS(for worms) |
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