Fig. 26.

Schematic representation of the construction of self-cleaving fusion systems. Filled triangle indicates cleavage sites and X stands for any AA. a The construct of the original C-terminal intein fusion in which the target protein is fused to the N-terminus of the CBD-tagged intein. b The SrtA fusion construct that contains an N-terminal affinity-tag, SrtA catalytic core, the LPXTG motif and the target protein. Cleavage at the LPXTG site allows the release of the target protein with an extra N-terminal glycine. c The FrpC fusion construct that consists of the target protein and the affinity-tagged SPM. Cleavage at the Asp–Pro site (the first two AAs of SPM) results in the release of the target protein with an extra aspartate residue at its C-terminus. d The CPD fusion construct in which the affinity-tagged CPD is fused to the C-terminus of the target protein. The VD double residue in the linker sequence comes from the SalI restriction site used for cloning whereas ALADGK are residues contained within the CPD. e The dithiocyclopeptide linker with one protease-sensitive site. The fusion protein is linked via a dithiocyclopeptide linker containing a thrombin-specific sequence, PRS. The design of dithiocyclopeptide linker was based on the structure of the cyclopeptide, somatostatin, with the replacement of AA residues 8–10, WKT, by a thrombin-specific cleavage sequence, PRS. f The dithiocyclopeptide linker with three secretion signal processing protease-sensitive sites. The fusion protein is linked via a dithiocyclopeptide linker containing Kex1, Kex2 and Ste13-specific cleavage sequences. Kex2 cleaves RR↓E. Kex1 and Ste13 remove C-terminal RR and N-terminal EA, respectively