Immobilization Tags

To achieve a covalent immobilisation of the different enzymes to an agarose membrane we chose three different immobilisation tags. All of them consist of two peptide chains which specifically bind to each other in solution and yield a covalent bond. For immobilisation purposes we used the SpyTag, the SnapTag and a split intein engineered from gyrase b of Synechocystis spec. PCC6803.

SpyTag

The SpyTag-sequence was taken from the iGEM registry and consists of a small peptide chain, called SpyTag, and a bigger protein-like structure, called SpyCatcher. Both BioBricks were created by Team TU-Munich in 2013 and can be found under the identifiers BBa_K1159200 (SpyCatcher) and BBa_K1159201 (SpyTag). The short SpyTag polypeptide (AHIVMVDAYKPTK) was C-terminally fused with 6-aminohexanoic acid as a spacer, followed by a carboxyfluorecine labelled lysine residue and a cystein residue for binding of the SulfLink. With this we achieve a fluorescent marker in our peptide to yield an indicator for correct immobilisation of the SpyTag to the target protein or the matrix. Because of the cystein residue at the N-terminus of the SpyTag, it can be covalently bound to a matrix via sulfo-link (for further information about the SpyTag have a look at our page about synthetic peptides). In order to immobilize our enzymes, they were C-terminally fused to the coding region of the SpyCatcher, which could then bind to the SpyTag, attached to the matrix.

SnapTag

The SnapTag is originally a labelling system, used to tag Proteins with fluorescent molecules. It is a derivate from human O-6-alkylguaninalkyltransferase. In our system the same principle is used, but instead of attaching a fluorescent marker, we attach the Snap-protein to a membrane coated in Snap-substrate. The Snap-protein itelf is a protein chain (182 amino acids), C-terminally fused to the target protein. The membrane is coated in the so called Snap-substrate, a 1-benzylguanidine. The mechanism for covalent fusion of the two parts is a S_N2-reaction where the guanine is substituted by a cystein residue near the N-terminus of the attached protein, which forms a thioester bond.

Split Intein (Ssp gyrase b intein)

Intein immobilisation (Figure 1) has a significant difference to the other two tag systems: While in the other cases two peptide chains are connected by the tag, the intein itself isn’t present in the immobilised state anymore, thereby forming a direct connection. Artificially created split inteins are modified parts of natural occuring inteins - protein regions which cut themselves out of a protein chain via self-catalytic activity- that are cut in two pieces. If C-terminal and N-terminal part find eachother in solution under specific conditions (in our case the optimal conditions are room temperature and a pH value around 7.5) the intein’s highly specific self-splicing activity is induced and the intein then cuts itself out of the protein chain, leaving the regions around the recombined intein covalently bound to each other. The cleavage mechanism occurs by conserved cystein and asparagine resiudes at the ends of the intein region. For our system we chose a split intein from gyrase b of Synechocystis spec. PCC6803. We chose it in particular because of it’s published high binding efficiency (89%) and it having a very small C-split-intein (modified the ensure a working fluorecin test) (GVPVHNGA[6-AHS]K(Fl)C) which could be aquired by peptide synthesis in the lab, thus allowing the same modifications we also used for Spy-Tag. The N-terminal split-intein (~ 500 base pairs) was added to the C-terminus of the protein coding DNA region. Ssp Gyrase b N-split-intein was sent in to be part of the iGEM registry and can be found under the BioBrick-Number BBa_K1483003.

Figure 1: Schematic illustration of split intein function.