Team:Tuebingen/Project/SyntheticPeptides

From 2014.igem.org


Synthetic Peptides

Two of the previously described protein tags (SpyTag and Intein) can be used with synthetic peptides for covalent immobilization. First the peptides need to be bound to a matrix. In a second step they interact specifically with their corresponding protein-tag, thereby forming a covalent bond. Afterwards, the matrix can be loaded onto columns in order to modify solutions passing it. The covalent binding of tag and peptide ensures that the protein can not be washed from the column and is essential for systems, where the immobilized protein should not be detectable in the product solution.

The peptides consist of a tag-specific and an immobilization region. The tag-specific region is specific for the reactivity of the peptide with its corresponding protein tag.

The synthetic intein consists of 6 amino acids (GVFVHN) and was for purposes of better steric accessibility extended at the C-terminus by the two amino acids G and A generating the desired active GVFVHNGA peptide of the intein. This synthetic peptide interacts with the intein part of our fusion proteins which leads to an event similar to splicing. For a detailed mechanism visit the wiki-page concerning Protein-Tags.

The synthetic SpyTag consists of 13 amino acids. The sequence did not require additional modification since it was proven by Zakeri et al. in 2011 to be suitable for our needs. The resulting functional sequence of the SpyTag is therefore AHIVMVDAYKPTK. The reactivity of this part is mediated by the aspartic acid at position 7, which forms an iso-peptide-bond with a corresponding lysine residue in the SpyCatcher.

The immobilization-region is designed for the binding to the solid support and detection of the peptides. It is located at the C-terminus of the tag-specific region and consists of aminocaproic acid (ε-AHX or Ahx), lysine and cysteine. The ε-amino group of the lysine is coupled to a carboxyfluorescein to enable easy detection the peptide. In this way also the detection of our fusion proteins by fluorescence labeling is achievable with low effort. The ε-AHX functions as spacer between the variable region and the fluorescein to ensure that the reactivity is not decreased due to steric hinderance by the carboxyfluorescein. The cysteine at the C-terminus is used for immobilization on commercial available SulfoLink-Resins or other thio-reactive matrices. The sequence of the immobilization-region of our peptides is therefore AhxK*C, for K* as the labelled lysine.

In essence, the following sequences result from these considerations:

Synthetic Intein:  GVFVHNGA- AhxK*C
Synthetic SpyTag:  AHIVMVDAYKPTK- AhxK*C

Figure 1: Structure of our synthetic intein.

 

Figure 2: Structure of our synthetic SpyTag.

Click here in order to have a look at our protocol for the creation of synthetic peptides.