Characterization of a Naturally Occurring Trans-Splicing Intein from Synechocystis sp. PCC6803

A naturally occurring trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) was used to characterize the intein-catalyzed splicing reaction. Trans-splicing/cleavage reactions were initiated by combining the N-terminal splicing domain of the Ssp DnaE intein containing five native N-extein residues and maltose binding protein as the N-extein with the C-terminal Ssp DnaE intein splicing domain (EC) with or without thioredoxin fused in-frame to its carboxy terminus. Observed rate constants (k obs) for dithiothreitol-induced N-terminal cleavage, C-terminal cleavage, and trans-splicing were (1.0 ± 0.5) × 10-3, (1.9 ± 0.9) × 10-4, and (6.6 ± 1.3) × 10-5 s-1, respectively. Preincubation of the intein fragments showed no change in k obs, indicating association of the two splicing domains is rapid relative to the subsequent steps. Interestingly, when EC concentrations were substoichiometric with respect to the N-terminal splicing domain, the levels of N-terminal cleavage were equivalent to the amount of EC, even over a 24 h period. Activation energies for N-terminal cleavage and trans-splicing were determined by Arrhenius plots to be 12.5 and 8.9 kcal/mol, respectively. Trans-splicing occurred maximally at pH 7.0, while a slight increase in the extent of N-terminal cleavage was observed at higher pH values. This work describes an in-depth kinetic analysis of the splicing and cleavage activity of an intein, and provides insight for the use of the split intein as an affinity domain.