Difference between revisions of "Part:BBa K1965032"

 
 
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<partinfo>BBa_K1965032 short</partinfo>
 
<partinfo>BBa_K1965032 short</partinfo>
  
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description
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<h3>Introduction</h3>
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<p>The split protein system based on inducible dimerization is an attractive method to regulate protease activity. Wehr et al. <sup>[1]</sup>  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) <sup>[1]</sup> .</p>
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<p> Soybean mosaic virus protease (SbMVp) is a 27kDa protease from soybean mosaic virus (SbMV). SbMVp is also known as nuclear inclusion a protein (NIa) and is one of the three viral proteases responsible for the processing of the viral polyprotein to at least ten functional segments<sup>[2,3]</sup>. SbMVp has been recently studied by Seo et al. <sup>[3]</sup> as a tool for protein-protein interaction studies in the soybean. We converted SbMVp to split protease by splitting it at positions corresponding to the position of the previously described split TEV protease. </p>
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<p> FKBP (FK-506 binding protein) is a protein that binds small molecule rapamycin with high affinity. In combination with 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 (<a href="http://2016.igem.org/Team:Slovenia/Protease_signaling/Split_proteases">CID</a>). </p>
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<p>SbMVp has well defined seven amino acid recognition motif SbMVs which is dermined by amino acid sequence ESVSLQ-S. For a detailed description of SbMVp click <a href="https://parts.igem.org/Part:BBa_K1965040">BBa_K1965040</a>. </p>
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<h3>Characterization</h3>
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<p> This part consist of the C-terminus of soybean mosaic virus protease (SbMVp) fused to the FK-506 binding protein (FKBP) and works in combination with the part FRB:nSbMVp (<a href="https://parts.igem.org/Part:BBa_K1965033">BBa_K1965033</a>). </p>
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        <figcaption><b> Activitiy of split SbMVs based on rapamycin inducible system.</b><br/> HEK293T cells were trasnfected with 100 ng of the cycLuc_SbMVs reporter and 70 ng of each split SbMV fragment. The whole SbMVp (70 ng) was used as positive control. An increase in luciferase activity was detected in cells induced with rapamycin.
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<p> We tested rapamycin inducible split SbMVp system by measuring activity with the <a href=" http://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters "> cycLuc reporter </a>. Increasing luciferase activity was detected, correlating with the amount of the transfected protease fragments in stimulated cells <ref>1</ref>. 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.</p>
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<h3>References:</h3>
 +
 
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<sup>[1]</sup>Wehr, M. C. et al. Monitoring regulated protein-protein interactions using split TEV. Nat. Methods 3, 985–93 (2006).<br>
 +
<sup>[2]</sup>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). <br>
 +
<sup>[3]</sup>Seo, J.-K. et al. Engineering of soybean mosaic virus as a versatile tool for studying protein–protein interactions in soybean. Sci. Rep. 6, 22436 (2016). <br>
 +
<sup>[4]</sup>Banaszynski, L. A., Liu, C. W. & Wandless, T. J. Characterization of the FKBP‚Rapamycin‚FRB Ternary Complex. doi:10.1021/ja043277y<br>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 17:25, 18 October 2016


FKBP:cSbMVp:HA

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) [1] .

Soybean mosaic virus protease (SbMVp) is a 27kDa protease from soybean mosaic virus (SbMV). SbMVp is also known as nuclear inclusion a protein (NIa) and is one of the three viral proteases responsible for the processing of the viral polyprotein to at least ten functional segments[2,3]. SbMVp has been recently studied by Seo et al. [3] as a tool for protein-protein interaction studies in the soybean. We converted SbMVp to split protease by splitting it at positions corresponding to the position of the previously described split TEV protease.

FKBP (FK-506 binding protein) is a protein that binds small molecule rapamycin with high affinity. In combination with 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).

SbMVp has well defined seven amino acid recognition motif SbMVs which is dermined by amino acid sequence ESVSLQ-S. For a detailed description of SbMVp click BBa_K1965040.

Characterization

This part consist of the C-terminus of soybean mosaic virus protease (SbMVp) fused to the FK-506 binding protein (FKBP) and works in combination with the part FRB:nSbMVp (BBa_K1965033).

Activitiy of split SbMVs based on rapamycin inducible system.
HEK293T cells were trasnfected with 100 ng of the cycLuc_SbMVs reporter and 70 ng of each split SbMV fragment. The whole SbMVp (70 ng) was used as positive control. An increase in luciferase activity was detected in cells induced with rapamycin.

We tested rapamycin inducible split SbMVp 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.

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]Seo, J.-K. et al. Engineering of soybean mosaic virus as a versatile tool for studying protein–protein interactions in soybean. Sci. Rep. 6, 22436 (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 325
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]