Team:Heidelberg/pages/Circularization Constructs

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Revision as of 11:17, 17 October 2014

Introduction

The most promising approaches to circularize proteins are protein trans-splicing using split inteins [1] and Sortase A-catalyzed cyclization [3]. Both methods require the addition of specific proteins domains or peptides to the protein to be circularized. Consequently, on DNA level, creating circular proteins equals creating fusion proteins. However, existing protein fusion standards like [http://parts.igem.org/Help:Standards/Assembly/RFC23 RFC[23]] cause scars. Those scars on protein level may affect protein function and further complicate 3D-structure modeling. Therefore, we decided to create a new [RFC] that allows scarless cloning of inteins. Our intein circularization constructs apply to this standard, while our sortase constructs are closely related and can be used similarly. Detailed instructions on how to use our constructs are provided in our Toolbox Guide.

NpuDnaE intein RFC [i] circularization constructs

Figure 1) BBa_K1362000

NpuDnaE intein RFC [i] circularization construct

Figure 2) BBa_K1362001

NpuDnaE intein RFC[i] circularization construct (with FLAG and Smt3)

Design

Usage and Biology

Experience

Results

Sortase A circularization constructs

Figure 3) BBa_K1362002

Sortase A circularization construct (with His6)

Figure 4) BBa_K1362003

Sortase A circularization construct (with Smt3 and His6)

Design

Usage and Biology

mechanismus?

References

[1] Iwai, H., Lingel, a & Pluckthun, a. Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus. J. Biol. Chem. 276, 16548–54 (2001).

[2] Zettler, J., Schütz, V. & Mootz, H. D. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett. 583, 909–14 (2009).

[3] Antos, J. M. et al. A straight path to circular proteins. J. Biol. Chem. 284, 16028–36 (2009).