Part:BBa_K1638035
hTrx-based scaffold fused to T18 through intein and a flexible linker
This composite part consists of the hTrx-scaffold (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 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.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1563
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1229
Illegal BamHI site found at 711
Illegal XhoI site found at 2374 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
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