Difference between revisions of "Part:BBa K1638014"

Revision as of 13:54, 14 August 2015

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. 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) [1]. The scaffold is based on the human thioredoxin 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 Gly ³³ and Pro ³⁴ with Leu ³³ and Glu ³⁴ . The removal of the two cysteins at active site prevents the multimerization of the expressed proteins and induces flexibility of the inserted peptide aptamer. Borghouts C et al. experienced higher yields when the three cysteins: Cys ⁶² , Cys ⁶⁹ , Cys ⁷³ 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.

Sequence and Features

References

[1] 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.