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Latest revision as of 17:12, 18 October 2016


FKBP:cPPVp

Introduction

The split protein system based on inducible dimerization is an attractive method to regulate protease activity. Wehr et al. [4] described a split tobacco etch virus protease (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) [1] .

Plum pox virus protease (PPVp) is a protease from plum pox virus (PPV). PPVp is also known as the nuclear inclusion a protein (NIa) and is one of the three viral proteases responsible for the processing of the viral polyprotein to functional segments [1,3]. PPVp is one of the most studied potyviral proteases after TEVp. We converted PPVp to split protease by splitting it at a position corresponding to the position of the previously described split TEV protease.

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 [2] ( CID ).

PPVp has a well-defined seven amino acid recognition motif PPVs which is determined by the amino acid sequence NVVVHQ-A. For a detailed description of PPVp click BBa_K1965025.

Characterization

This part consist of the C-terminus of plum pox virus protease (PPVp) fused to the FK-506 binding protein (FKBP) domain and works in combination with part FRB:nPPVp (BBa_K1965027).

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

We tested rapamycin inducible split PPVp system by measuring activity with the cycLuc reporter (BBa_K1965042). 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.

References:

[1]Adams, M. J., Antoniw, J. F. & Beaudoin, F. Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Mol. Plant Pathol. 6, 471–87 (2005).
[2]Banaszynski, L. A., Liu, C. W. & Wandless, T. J. Characterization of the FKBP‚Rapamycin‚FRB Ternary Complex. doi:10.1021/ja043277y
[3]Garcia, J. A. & Lain, S. Proteolytic activity of plum pox virus—tobacco etch virus chimeric NIa proteases. FEBS Lett. 281, 67–72 (1991).
[4]Wehr, M. C. et al. Monitoring regulated protein-protein interactions using split TEV. Nat. Methods 3, 985–93 (2006).


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 BsaI.rc site found at 361
    Illegal BsaI.rc site found at 541