Part:BBa_K1202006

TAT(Trans-Activator of Transcription) protein

Trans Activator of Transcription (TAT) peptide is a special signal peptide that directs the attached proteins into eukaryotic cells in the extracellular matrix. The proteins conduct this kind of activity called "cell penetrating peptides" (CPP).

This allow the transportation of desired proteins into the cells. The efficiency of the part can be manipulated by adding additional sequences such as HA2.

In our project, we want to use this protein to direct our cancer killing protein "apoptin" into the colon cancer cells, efficiently.

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
  COMPATIBLE WITH RFC[1000]

Usage and Biology by iGEM Tuebingen 2019

Due to their penetrating activity, Cell penetrating peptides can be utilised as carriers of small therapeutic macromolecules or nucleic acids across membranes (Kamei et al.). Since cytotoxic effects have occured, induced by the disintegration of the plasma membrane, improvement and development of cell penetrating peptides is necessary.

Kamei et al. propose that cell penetrating peptides gain their activity through the abundance of multiple positive charges (argine, R) and the ability to form an amphipathic helix.

Tat is commonly used to deliver DNA across membranes in order to overcome the issues proposed by low transfection efficiency in cell culture (Saleh et al.). It consists of up to 35 amino acids of the HIV-1 full length protein Tat (Wender et al.). Its penetrating acitivity is supposed to be induced by guanidinium groups (Wender et al.)

Saleh et al. showed that Tat's penetrative activity can be significantly increased upon the covalent attachment of LK-15 to Tat. Moreover, they demonstrated that Tat-LK15/DNA complex treatment results in stronger leakage of the membrane, which could be causing the increased transfection activity (Salah et al.).

Improvement of Tat (BBa_K1202006) by iGEM Tuebingen 2019

To improve Tat (BBa_K1202006), iGEM Team Tuebingen has hereby introduced Tat-LK15 to the iGEM registry. According to Saleh et al. DNA complexes with Tat–LK15 confer to a higher transfection efficiency than Tat. This can be confirmed by our software tool C3Pred (see below).

The improved activity of TAT-LK15 over Tat was confirmed by our CPP transport activity prediction tool C3Pred. TAT-LK15 scored significantly higher results than the wildtype Tat. This can partly be explained by the presence of multiple positively charged residues in the LK15 peptide, which have been shown to be associated with high transport efficiency by our software tool.

Our tool shows that the penetrative activity of Tat is about 5.5, while Tat-LK15 has an activity of 7.8, an improvement by approx. 42% (see Table).

To use the tool and to get more information on how it operates, please visit the wiki of iGEM Team Tübingen 2019.

Biosafety

Together with professional safety officials, Team iGEM Tuebingen 2019 has evaluated the biosafety of cell penetrating peptides and has come to the conclusion that cell penetrating peptides are to be classified as BSL1.

References

1. Saleh, Amer F., et al. "Improved Tat-mediated plasmid DNA transfer by fusion to LK15 peptide." Journal of Controlled Release 143.2 (2010): 233-242.
2. Kamei, Noriyasu, et al. "Usefulness of cell-penetrating peptides to improve intestinal insulin absorption." Journal of Controlled Release 132.1 (2008): 21-25.
3. Paul A. Wender, Dennis J. Mitchell, Kanaka Pattabiraman, Erin T. Pelkey, Lawrence Steinman, Jonathan B. Rothbard "The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters". Proceedings of the National Academy of Sciences Nov 2000, 97 (24) 13003-13008; DOI: 10.1073/pnas.97.24.13003