Part:BBa_K1965036

Myc:FRB:nSuMMVp

Introduction

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

Sunflower mild mosaic virus protease (SuMMVp) is a protease from sunflower mosaic virus (SuMMV). SuMMVp 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 into at least ten functional segments^[2,3]. SuMMVp has been used by Fernandez-Rodriguez et al. ^[3] in a study of genetic circuits using potyviral proteases. We converted SuMMVp to a 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 ( CID ). ^[4]

SuMMVp has a well-defined seven amino acid recognition motif SuMMVs which is determined by the amino acid sequence EEIHLQ-S/G. For a detailed description of SuMMVp click BBa_K1965041

Characterization

This part consist of the N-terminus of sunflower mild mosaic virus protease (SuMMVp) fused to the FKBP-rapamycin binding (FRB) domain and works in combination with the part FKBP:cSuMMVp (BBa_K1965035).

We tested the rapamycin inducible split SuMMVp 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^[4].

Figure 1: Activity of split proteases based on rapamycin inducible system
HEK293T cells were transfected 100 ng of the cycLuc_SuMMVs reporter and 70 ng of each split SuMMVp fragment. The whole SuMMVp (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] 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).
^[3] Fernandez-Rodriguez, J. & Voigt, C. A. Post-translational control of genetic circuits using Potyvirus proteases. Nucleic Acids Res. 44, 6493–502 (2016).
^[4] 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 313
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]