Part:BBa_K1638035

hTrx-based scaffold fused to T18 through intein and a flexible linker

This composite part consists of the hTrx-scaffold [1] (used for presenting peptide aptamers) fused to the T18 domain of CyaA (used in the bacterial two-hybrid system) through intein and a linker. This part is used in the bacterial two-hybrid screening [2] for peptide aptamers against specific targets. When a target candidate is fused to the T25 domain of CyaA, and a peptide aptamer binds the target, the T18 and T25 domain is brought into proximity of one another and the formation of cAMP is induced. When combined with a cAMP reporter system, like a system promoting the transcription of RFP, the presence of red-flourescent bacteria indicates an interaction between the peptide aptamer and the target protein.

Intein is added in-between the T18 domain and the hTrx-based peptide apatamer for affinity purification of the hTrx-based peptide aptamer. Intein is a thiol-induced self-cleavable protein that enables the release of the C-terminal fused hTrx-based peptide aptamer [3].

The Library

In oder to use this part for peptide aptamer screening, a random nucleotide library has to be inserted into the scaffold. This library codes for a random peptide library that enables screening of a diverse set of peptide aptamers with different specificity. Typically, the library consist of 20 sets of the codon NNK (where N is A,T,G or C and K is G or C). See design for further notes on design of library.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1563
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1229
  Illegal BamHI site found at 711
  Illegal XhoI site found at 2374
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 252
  Illegal NgoMIV site found at 662
  Illegal NgoMIV site found at 2106
  Illegal AgeI site found at 468
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1]: Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(10):5752-6.
[2]: Borghouts C, Kunz C, Delis N, Groner B. Monomeric Recombinant Peptide Aptamers Are Required for Efficient Intracellular Uptake and Target Inhibition. Molecular Cancer Research. 2008;6(2):267-81.
[3]: IMPACT™ Kit. 2015. New England Biolabs; [accessed 2015 Sep 11]. https://www.neb.com/products/e6901-impact-kit