Part:BBa_K1638014

hTrx scaffold for peptide aptamers with 3xFLAG-tag

This part is a human thioredoxin-based scaffold for the presentation of peptide aptamers. The hTrx-scaffold contains a xhoI restriction site that enables insertion of a random DNA library. A 3xFLAG-tag is added to the C-terminal end of the scaffold. This affinity tag can be used for detection and/or purification purposes.

An antibody-mimetic

Peptide aptamers are combinatorial recognition proteins that provide high specificity and strong binding affinity (typically in the range, 10 nM-100 nM) [1,2]. They consist of a variable peptide sequence inserted into a protein scaffold. This variable peptide loop makes up a binding domain that enables binding to various proteins.

The design of this peptide aptamer scaffold is inspired by (Borghouts C et al., 2008) [3]. The scaffold is based on the human thioredoxin molecule that has been modified by the replacement of five cysteins with two glycins and three serines. This includes two cysteins in the active site. The mutations is listed below:

• C32>G
• C35>G
• C62>S
• C69>S
• C73>S

Additionally a xhoI restriction site (CTCGAG) was introduced to the active site, replacing Gly³³ and Pro³⁴ with Leu³³ and Glu³⁴. The removal of the two cysteins at the active site prevents the multimerization of the proteins and induces flexibility of the inserted peptide aptamer allowing them to adopt various conformations. Borghouts C et al. experienced higher yields when the three cysteins: Cys⁶², Cys⁶⁹, Cys⁷³ was removed. For this reason, these cysteins was replaced with serines. In order to optimize the expression of our scaffold in E. coli, the sequence was also codon-optimized for use in a E. coli K12 strain.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 100
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] Colas P, Cohen B, Jessen T, Grishina I, McCoy J, Brent R. Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2. Nature. 1996;380(6574):548-50.

[2] Seigneuric R, Gobbo J, Colas P, Garrido C. Targeting cancer with peptide aptamers. Oncotarget. 2011;2(7):557-61.

[3] 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.