Part:BBa_K1965038

FKBP:cTEV

Introduction

The split protein system based on inducible dimerization is an attractive method to regulate protease activity. Wehr et al. ^[1] 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.

FKBP is a protein that binds the small molecule rapamycin with high affinity. In combination with the FKBP-rapamycin binding (FRB) domain 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 C-terminus of the tobacco etch virus protease (TEVp) fused to the FK-506 binding protein (FKBP) and works in combination with the part FRB:nTEVp (BBa_K1965039).

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].

Activity of split proteases based on rapamycin inducible system.
HEK293T cells were transfected 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

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

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 325
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 646