Fig. 3. Simplification of enzymatic head-to-tail cyclisation using autocyclases.

a A flowchart outlining each distinct step in the process, comparing autocyclisation with the previously described bimolecular reaction⁸. Preparation step 1 (Prep. 1) differs between the two methods. In the unimolecular reaction, this step involves dialysis of the fusion protein into a suitable solution for TEV cleavage and cyclisation reactions, which are performed as a single step (5). In the bimolecular reaction, this step (4) includes the TEV cleavage reaction as well as a number of buffer exchange steps (preceding and following this step). Following reverse IMAC purification (IMAC 2), the sample can be loaded directly onto an ion exchange (IEX) column without the previously described buffer exchange step (Prep. 2). b cMSP11 cyclisation using the proposed method, showing SDS-PAGE lanes corresponding to start and end of steps 5 and 6.