Part:BBa_K1965039

FRB:nTEVp

Introduction

The split protein system based on inducible dimerization is an attractive method to regulate protease activity. Wehr et al.^[2] described a split TEVp expressed as two functionally inactive fragments; the N-terminal (1 – 118 aa) and C-terminal (119 – 242 aa) protease fragments (referred to as cTEVp and nTEVp). When the two fragments were coexpressed as fusion constructs with adjacent dimerization partners, the split TEVp was able to reconstitute and regain its catalytic activity, demonstrating that the activity of split TEVp could be controlled through ligand induced protein – protein interactions.

FRB is a protein that binds the small molecule rapamycin with high affinity. In combination with the FK-506 binding protein (FKBP) it is widely used for induced dimerization of proteins. Proteins of interest can be fused to FKBP or FRB and then conditionally dimerized by the addition of rapamycin (CID).^[2].

TEVp has a well-defined seven amino acid recognition motif TEVs which is determined by the amino acid sequence ENLYFQ-G/S. For a detailed description of TEVp click BBa_K1965009.

Characterization

This part consist of the N-terminus of tobacco etch virus protease (TEVp) fused to the FKBP-rapamycin binding (FRB) domain and works in combination with the part FKBP:cTEVp (BBa_K1965038).

Activity of split proteases based on rapamycin inducible system
HEK293T cells were transfected with 100 ng of the cycLuc_TEVs reporter and 70 ng of each split TEVp fragment. The whole TEVp (70 ng) was used as positive control. An increase in luciferase activity was detected in cells induced with rapamycin.

We tested the rapamycin inducible split TEVp system by measuring activity with the cycLuc reporter. Increasing luciferase activity was detected correlating with the amount of the transfected protease fragments in stimulated cells (1). Luciferase in unstimulated cells remained inactive even at the highest amount of transfected protease fragments, proving low leakage and high inducibility of the split protease system in response to rapamycin^[2].

Improvement by UPenn 2022

This part (along with BBa_K1965038) was made optogenetic by adding an iLID-sspB system. Optogenetics is a powerful research tool for controlling cell behavior that offers precise spatiotemporal control by varying the wavelength of lights used. The spatial precision can be achieved at the level of micrometers while the temporal precision can be achieved at <1ms^[3]. This light input generates a response in photosensitive proteins bound to target proteins, which allows researchers to control different aspects of cell activity. iLID (improved light-induced dimer) and sspb (stringent starvation protein B) are a pair of heterodimerizing proteins that reversibly bind under 450 nm light within seconds of exposure. iLID consists of a bacterial SsrA (small stable RNA A) peptide embedded in the naturally occurring photoswitch AsLOV-2 (Avena sativa light-oxygen voltage 2 domain)^[4]. Before light activation the SsrA is sterically blocked from its binding partner sspb by AsLOV-2 domain however upon light exposure the domain undocks and heterodimerization takes place. Optogenetic heterodimerizing proteins are very useful in biological signaling studies because they allow cellular localization of POIs. For example iLID has already been applied in reversible controlling small GTPase signaling, which are enzymes that regulate cytoskeletal reorganization, cell polarity, cell cycle progression, gene expression and many other significant events in cells, such as the interaction with foreign particles^[5]. The improved part (BBa_K4444017) replaced FRB with iLID and added mCherry and an ER localization tag. Additionally, FKBP was replaced with sspB-BFP in BBa_K4444018 (which is a modification of BBa_K1965038). The system is kept in the ER due to the localization tag (which can be seen under the microscope due to mCherry) and, when activated under blue light, the iLID and sspB will dimerize bringing the two TEVp fragments together. By making these parts optogenetic, we greatly improve the precision of spacial and temporal control.

Design of iLID-nTEV system

References

^[1]Wehr, M. C. et al. Monitoring regulated protein-protein interactions using split TEV. Nat. Methods 3, 985–93 (2006).
^[2]Banaszynski, L. A., Liu, C. W. & Wandless, T. J. Characterization of the FKBP‚Rapamycin‚FRB Ternary Complex. doi:10.1021/ja043277y
^[3]Zhu, Danqing, et al. “Optogenetic Application to Investigating Cell Behavior and Neurological Disease.” Frontiers in Cellular Neuroscience, vol. 16, 22 Feb. 2022, 10.3389/fncel.2022.811493.
^[4]Guntas, Gurkan, et al. “Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins.” PNAS, vol. 112, no. 1, 2014, pp. 112-117, https://doi.org/10.1073/pnas.1417910112.
^[5]Song, Siyang, et al. “Small GTPases: Structure, Biological Function and Its Interaction with Nanoparticles.” Asian Journal of Pharmaceutical Sciences, vol. 14, no. 1, Jan. 2019, pp. 30–39, www.sciencedirect.com/science/article/pii/S1818087618304641, 10.1016/j.ajps.2018.06.004.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 283
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 610