Part:BBa_K3096016

Tat-LK15 - improved Cell penetrating peptide

TAT-LK15 is a short fusion peptide of the cell-penetrating peptide (CPP) Tat (transcactivator of transcription) and the synthetic LK15 peptide. Adding LK15 to Tat results in improved transport efficiency across the cell membrane. The part has been reported to be able to transport nucleic acids through membranes, which allows for novel transfection protocols (Saleh et al.). LK15 consists of 15 amino acids, which are all either leucine (L) or lysine (K) residue, resulting in an amphipathic protein (Saleh et al.).

Sequence and Features

Tat: RKKRRQRRRGGG-CONH2 LK15: KLLKLLLKLLLKLLK-COOH Tat-LK15: RKKRRQRRRGGGKLLKLLLKLLLKLLK-CONH2

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]

Biology & Usage

Through 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, induced by the disintegration of the plasma membrane, have occured, improvement and development of cell penetrating peptides is necessary.

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

Improvement of Tat (BBa_K1202006) by iGEM Tuebingen 2019

To improve Tat (BBa_K1202006), added to the iGEM registry by iGEM Team, 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).

Characterisation of Tat-LK15

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 associated with high transport efficiency by our software tool.

As can be seen in the Table, 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%.

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