Ubiquiton Kits
(Kit #
1000000242, 1000000243, and 1000000244
)
Depositing Lab: Helle Ulrich
The Ubiquiton system allows the inducible, linkage-specific polyubiquitylation of a protein of interest, accommodating for linear (M1-), K48-, and K63-linkage.
Kit #1000000242 Ubiquiton In Vitro Kit: This kit contains plasmids for the production of custom linkage-specific ubiquitin protein ligases (E3s) and appropriately tagged substrate proteins of interest for in vitro experiments. The kit also includes plasmids for positive and negative controls to ensure linkage-selective modification.
Kit #1000000243 Ubiquiton Yeast Kit: This kit contains plasmids for the expression of custom linkage-specific ubiquitin protein ligases (E3s) and for introducing appropriate ubiquitin acceptor tags to the substrate proteins of interest, either by PCR-based tagging or vector-based cloning, in budding yeast. The kit also includes plasmids for positive and negative controls to ensure linkage-selective modification, variations of selectable markers (URA3, LEU2, hygromycin), detection tags (GFP, mCherry, myc, His6), and the option to use nuclear localization signals on the E3s.
Kit #1000000244 Ubiquiton Human Kit: This kit contains plasmids for the expression of custom linkage-specific ubiquitin protein ligases (E3s) and appropriately tagged substrate proteins of interest in human cells. The kit also includes plasmids for positive and negative controls to ensure linkage-selective modification, overexpression of relevant ubiquitin conjugating enzymes (E2s), and a selection of detection tags (GFP, mCherry, FLAG, His6).
These kits will be sent as bacterial glycerol stocks in 96-well plate format.
Ordering
| Item | Catalog # | Description | Quantity | Price (USD) | |||
|---|---|---|---|---|---|---|---|
| Kit | 1000000242 | Ubiquiton In Vitro Kit | 1 | $400 | Add to Cart | ||
| Kit | 1000000243 | Ubiquiton Yeast Kit | 1 | $400 | Add to Cart | ||
| Kit | 1000000244 | Ubiquiton Human Kit | 1 | $400 | Add to Cart | ||
Original Publication
Ubiquiton—An inducible, linkage-specific polyubiquitylation tool. Renz C, Asimaki E, Meister C, Albanèse V, Petriukov K, Krapoth NC, Wegmann S, Wollscheid HP, Wong RP, Fulzele A, Chen JX, Léon S, Ulrich HD. Mol Cell. 2024 Jan 18;84(2):386-400.e11. doi: 10.1016/j.molcel.2023.11.016. PubMed (Link opens in a new window) Article (Link opens in a new window)
Description
The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse functionality. While numerous tools exist to decipher the anatomy of ubiquitin structures, such as linkage-specific antibodies or affinity probes, controlling linkage-specific polyubiquitylation of a protein of interest in cells or in vitro is still a challenge. Here we introduce the "Ubiquiton" system, a set of engineered ubiquitin protein ligases (E3s) and matching ubiquitin acceptor (Ubo-) tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins. Application of this research tool facilitates investigating the function of protein polyubiquitylation by separating the consequences of a ubiquitylation event from the signal that normally induces it or by altering the linkage of a polyubiquitin chain in a controlled manner. Moreover, it allows for the targeted polyubiquitylation of proteins that are not normally subject to such modification.
The Ubiquiton system is based on a set of linkage-specific E3s capable of extending a ubiquitin unit to a polyubiquitin chain with a specified linkage (M1, K48, or K63). They are recruited to the substrate of interest via a rapamycin-inducible dimerization tag. An initiator ubiquitin is introduced by means of a split ubiquitin unit, assembled by half-ubiquitin modules on the E3 and the substrate, respectively. In this manner, the initiator ubiquitin forms upon rapamycin-induced assembly of the E3-substrate complex and polyubiquitin chain formation is initiated on the substrate Ubo-tag (Figure 1). As an important negative control for target specificity of the ubiquitylation reaction, each kit also contains plasmids for generating substrate mutants in which the acceptor residue (M1, K48, or K63) of the Ubo-tag is blocked or mutated.
Kit #1000000242: Ubiquiton In Vitro Kit
The kit contains a set of plasmids for production of custom E3s, their cognate ubiquitin-conjugating enzymes (E2s), and appropriately tagged substrate proteins of interest in E. coli (or in one case insect cells) for in vitro experiments. Researchers will use the vectors to a) clone their proteins of interest with a suitable Ubo-tag and b) produce recombinant custom ubiquitylation enzymes (E2s and E3s) for in vitro ubiquitylation assays. Plasmids for production of E1 and ubiquitin are not included. The kit also contains plasmids for Ubo-GFP as a positive control substrate.
Kit #1000000243: Ubiquiton Yeast Kit
The kit contains a set of plasmids for expression of custom E3s and for introducing appropriate Ubo-tags to the substrate proteins of interest in yeast. Researchers will use the vectors for a) PCR-based tagging or vector-based cloning to introduce Ubo-tags to their proteins of interest and b) expression of custom E3s to assess the in vivo modification of their proteins of interest. Included in the kit are plasmids for Ubo-GFP as a positive control substrate. Finally, the kit includes a range of selectable markers (URA3, LEU2, hygromycin), detection tags (GFP, mCherry, myc, His6), and the option to use nuclear localization signals on the E3s.
Kit #1000000244 Ubiquiton Human Kit
The kit contains a set of plasmids for expression of custom E3s and for introducing appropriate Ubo-tags to the substrate proteins of interest in human cells. Researchers will use the vectors to a) clone and express their proteins of interest with a suitable Ubo-tag and b) express custom E3s to assess the modification of these proteins in human cells. Included in the kit are plasmids for Ubo-GFP and Ubo-EGFR as positive control substrates. Finally, the kit includes a selection of detection tags (GFP, mCherry, FLAG, His6) and plasmids for overexpression of relevant ubiquitin-conjugating enzymes (E2s).
Figure 1: Ubiquiton (Ubo)-tags and enzymes for inducible M1-, K48-, and K63-linked polyubiquitylation. Note for M1-Ubiquiton, Ubo-E3(1) utilizes either human UBCH7 or a related yeast E2. For use of K48-Ubiquiton in human cells, Ubo-E3(48) requires co-expression of budding yeast Ubc7 as a cognate E2. For K63-Ubiquiton, Ubo-E3(63) is capable of utilizing yeast Ubc13 and Mms2 or their human homologs. Image modified from Renz et al. 2023, under a Creative Commons CC-BY-NC license.
How to Cite this Kit
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.
For your Materials and Methods section:
“The Ubiquiton Kit was a gift from Helle Ulrich (Addgene kit # ).”
For your Reference section:
Ubiquiton—An inducible, linkage-specific polyubiquitylation tool. Renz C, Asimaki E, Meister C, Albanèse V, Petriukov K, Krapoth NC, Wegmann S, Wollscheid HP, Wong RP, Fulzele A, Chen JX, Léon S, Ulrich HD. Mol Cell. 2024 Jan 18;84(2):386-400.e11. doi: 10.1016/j.molcel.2023.11.016. PubMed (Link opens in a new window) Article (Link opens in a new window)
Ubiquiton In Vitro Kit - #1000000242
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Ampicillin | |
| Kanamycin |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pET22b-His6-UBCH7 | Ampicillin |
| A / 2 | pFastBac1-His6-E3(1)-CUbo-TwinStrep | Ampicillin |
| A / 3 | pMAL-TEV2-Ubc7-His6 | Ampicillin |
| A / 4 | pET21(+)-His6-Ubc13 | Ampicillin |
| A / 5 | pET15b-His6-CUbo-GFP | Ampicillin |
| A / 6 | pET15b-His6-CUbo(K48R)-GFP | Ampicillin |
| A / 7 | pET15b-His6-CUbo(K63R)-GFP | Ampicillin |
| A / 8 | pET24a-NUbo-MCS-His6 | Kanamycin |
| A / 9 | pET24a-His7-NUbo-MCS | Kanamycin |
| A / 10 | pET24a-His6-CUbo-MCS | Kanamycin |
| A / 11 | pET24a-His6-CUbo(K48R)-MCS | Kanamycin |
| A / 12 | pET24a-His6-CUbo(K63R)-MCS | Kanamycin |
| B / 1 | pET24a-NUbo-E3(48)-His6 | Kanamycin |
| B / 2 | pET30-His6-TEV-Mms2 | Kanamycin |
| B / 3 | pET24a-NUbo-E3(63)-His6 | Kanamycin |
| B / 4 | pET24a-NUbo-GFP-His6 | Kanamycin |
| B / 5 | pET24a-His7-NUbo-GFP | Kanamycin |
Kit Plate Map - #1000000242
Ubiquiton Yeast Kit - #1000000243
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Ampicillin |
Inventory
Kit Plate Map - #1000000243
Ubiquiton Human Kit - #1000000244
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Ampicillin |