Overview¶
The ClpXP control module enables the ATP-dependent, targeted degradation of ssrA-tagged proteins McGinness, Baker, and Sauer, 2006. It is based on the complex formed by the AAA+ ATPase ClpX and the tetradecameric peptidase ClpP. The module can be implemented using purified protein, in situ expressed proteins from DNA templates, or combinations thereof.
Cartoon of the general mechanism of protein degradation by ClpXP, an ATP-dependent protease. Adapted from R. Wedam, et al.
| Name | Length (bp) | File |
|---|---|---|
pT7-ClpX | N/A | (upcoming) |
pT7-ClpP | N/A | (upcoming) |
pT7-deGFP-ssrA | N/A | (upcoming) |
Cytosols¶
Usage¶
The module can be implemented from purified proteins alone, from in situ expressed proteins encoded on DNA templates, or from combinations thereof.
Reaction Table 1. The control module implemented from purified proteins. Volumes in µL.
| Component | Sample 1 | Sample 2 | Sample 3 | Control |
|---|---|---|---|---|
| Purified deGFP-ssrA (41.2 µM) | 0.5 | 0.5 | 0.5 | 0.5 |
| Purified ClpP (79.9 µM) | 0.5 | 0.5 | 0 | 0 |
| Purified ClpX (53.7 µM) | 0.5 | 0 | 0.5 | 0 |
| NEB PURExpress Solution A | 4 | 4 | 4 | 4 |
| NEB PURExpress Solution B | 3 | 3 | 3 | 3 |
| RNase Inhibitor | 0.5 | 0.5 | 0.5 | 0.5 |
| Nucleus Free Water | 1 | 1.5 | 1.5 | 2 |
| Total | 10 | 10 | 10 | 10 |
Reaction Table 2. The control module implemented from in situ expressed proteins. Steady-state levels can be tuned by varying the concentration of in situ expressed ClpXP proteins. Volumes in µL.
| Component | Sample 1 | Sample 2 | Sample 3 | Sample 4 |
|---|---|---|---|---|
| pT7-deGFP-ssrA (63.5 ng/µL) | 0.5 | 0.5 | 0.5 | 0.5 |
| pT7-ClpP (17.5 ng/µL) | 0.4 | 0.4 | 0.6 | 0.8 |
| pT7-ClpX (17.5 ng/µL) | 0.4 | 0.4 | 0.6 | 0.8 |
| NEB PURExpress Solution A | 4 | 4 | 4 | 4 |
| NEB PURExpress Solution B | 3 | 3 | 3 | 3 |
| RNase Inhibitor | 0.5 | 0.5 | 0.5 | 0.5 |
| Nucleus Free Water | 1.6 | 1.2 | 0.8 | 0.4 |
| Total | 10 | 10 | 10 | 10 |
Expected Performance¶
Module performance in PURE is documented in the DevNote ClpXP Module Validation in PURE.
GFP fluorescence of samples containing purified proteins incubated at 37°C for 4 hours. These results correspond to Reaction Table 1.
GFP fluorescence signal produced using pT7-deGFP-ssrA DNA in PURE reactions incubated at 37°C for 6 hours. ClpX and ClpP DNAs are co-expressed in the same PURE reaction. These results correspond to Reaction Table 2.
Cells¶
Cell-context validation of the ClpXP module is documented in the DevNote ClpXP Module Validation in Cells.
The ClpXP Control Module in the context of the Developer Cell. Other Developer Cell Modules are grayed out.
- McGinness, K. E., Baker, T. A., & Sauer, R. T. (2006). Engineering Controllable Protein Degradation. Molecular Cell, 22(5), 701–707. 10.1016/j.molcel.2006.04.027
- Wedam, R., Greer, Y. E., Wisniewski, D. J., Weltz, S., Kundu, M., Voeller, D., & Lipkowitz, S. (2023). Targeting Mitochondria with ClpP Agonists as a Novel Therapeutic Opportunity in Breast Cancer. Cancers, 15(7), 1936. 10.3390/cancers15071936