Part:BBa_K1965025

PPVp

Introduction

Plum pox virus protease (PPVp) is a protease from the plum pox virus (PPV). PPVp is also known as the nuclear inclusion a protein (NIa) that is one of the three viral proteases responsible for processing of the viral polyprotein to functional segments ^[1,2] . PPVp is one of the most studied potyviral proteases after TEVp. Its substrate (PPVs) has an amino acid sequence NVVVHQ-A. In contrast to TEVp, it has been reported that PPVp is resistant to self-cleavage at the C-terminus ^[3,4].

Characterization

PPVp has a well-defined seven amino acid recognition motif PPVs, which is defined by the amino acid sequence NVVVHQ-A ^[3]. We included that sequence into the cyclic luciferase (cycLuc_PPVs) reporter construct.

PPVp construct was expressed under the CMV promoter. The coding sequence for PPVp was deposited in pSB1C3.

PPVp was used as one of the new orthogonal proteases in our protease based signaling platform. 1 shows that PPVp successfully cleaves its substrate and is orthogonal to other proteases (TEVp, SbMVp and SuMMVp).

Protease orthogonality.
HEK293T cells were transfected with 100ng of the indicated cycLuc reporters and 70ng of the indicated proteases. Luciferase activity was detected only in the presence of a protease and cycLuc reporter containing appropriate protease cleavage site.

References

^[1]Adams, M. J., Antoniw, J. F., & Beaudoin, F. (2005). Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Molecular Plant Pathology, 6(4), 471–487. https://doi.org/10.1111/j.1364-3703.2005.00296.x
^[2]Zheng, N., Pérez, J. de J., Zhang, Z., Domínguez, E., Garcia, J. A., & Xie, Q. (2008). Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease. Protein Expression and Purification, 57(2), 153–162. https://doi.org/10.1016/j.pep.2007.10.008
^[3]Garcia, J. A., & Lain, S. (1991). Proteolytic activity of plum pox virus-tobacco etch virus chimeric NI a proteases. FEBS Letters, 281(1–2), 67–72. https://doi.org/10.1016/0014-5793(91)80360-F

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 382
  Illegal BsaI.rc site found at 562