By O. Zerbe
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The first fragment was a fusion protein of the N-terminal domain of the split target protein and the N-terminal split intein-1. The second segment consisted of the C-terminal split intein-1 (PI-PfuI), the central part of the split target protein fragment and the N-terminal split intein-2 (PI-PfuII). The third segment contained the C-terminal split intein-2 fused to the C-terminal fragment of the split target protein. Ligation of the first and the second fragment was facilitated by intein-1, while the second and the third fragments were ligated by intein-2.
1. On the other hand, the split intein approach requires all four reaction steps. In this case the success of the reaction depends on refolding properties of the split intein (Sect. 2). 1 Intein-Mediated Protein Ligation (IPL)/Expressed Protein Ligation (EPL) using the IMPACT System The IMPACT (Intein-Mediated Purification with an Affinity Chitin-binding Tag) system was originally developed as a novel purification method by New England Biolab. It makes use of a modified intein, SceVMA, in which the active site was mutated from HisAsn-Cys to His-Ala-Cys, so that the usual cleavage due to succinimide formation involving the side-chain of Asn is no longer possible (Figs.
In addition, the stabilization of proteins without loss of function is not a trivial problem. One new approach to stabilizing proteins without changing the primary sequence is to introduce backbone cyclization . No mutations in the primary sequence are introduced by this method, although it might be necessary to insert a flexible linker comprising several residues to join the termini [74–76]. Polymer theory by Flory predicts an improvement in protein stability upon cyclization, because the entropy of the unfolded states should be reduced .