Difference between revisions of "Part:BBa K1483003"

Revision as of 01:31, 19 September 2015

Ssp GyrB Split Intein

N-Intein part of the ssp GyrB split intein. Can be used in conjunction with the 6 amino acid long C-Intein to N-terminally immobilize a peptide onto a matrix. For immobilisation, parts need to be fused to the N-terminus of the N-intein. The C-terminus of the C-Intein is fused the matrix. The split intein recombines and splices itself out, thereby specificially immobilising the N-terminus of the peptide to the matrix.

The sequence of the corresponding C-Intein is: H2N-GVPVHN-COOH (aquired by peptide synthesis and is thus not part of the registry)

This part can also be used in order to create fusion proteins, by attaching the C-Intein to a protein instead of the matrix. The part is therefore designed in RFC25.

Contribution by Team Tuebingen 2015:

We expressed the ssp GyrB split intein as a fusion protein with an enzyme weighing 52.2 kDa (BBa_K1483000) using the pETue vector (BBa_K1680026) in BL21(DE3). We were able to show that the protein can be expressed and does react with the synthetic peptide described in the design section. We were able to confirm this using a bandshift assay, which uses the intein's protein-splicing mechanism.

Expression: This part can be expressed by growing a culture transformed with the above mentioned plasmid to OD(600)=0.3, inducing with 2 mM IPTG and expressing over night at room temperature. The culture can then be lysed and the protein purified using the 6xHis-Tag. The result of such an experiment is shown below. The band at 60 kDa in the eluate fraction represents the NAGA-Intein fusion protein.

900px

To establish that the split intein interacts and is active in comination with the synthetic peptide described in the design section, we incubated the peptide and the purified protein for 48 hours at room temperature. In the gel shown below, after addition of the peptide, an additional band appears slighly below 60 kDa.

900px

The NAGA-Intein construct has either reacted in the predicted way, or a side reaction called N-Cleavage has occured. During N-Cleavage, the the N-Extein is cleaved from the intein without a splicing reaction taking place. For a discussion on this see our WIKI!

Sequence and Features