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Background  Protein  trans -splicing by naturally occurring split DnaE inteins is used for protein ligation of foreign peptide fragments. In order to widen biotechnological applications of protein  trans -splicing, it is highly desirable to have split inteins with shorter C-terminal fragments, which can be chemically synthesized.    Principal Findings  We report the identification of new functional split sites in DnaE inteins from  Synechocystis sp . PCC6803 and from  Nostoc punctiforme . One of the newly engineered split intein bearing C-terminal 15 residues showed more robust protein  trans -splicing activity than naturally occurring split DnaE inteins in a foreign context. During the course of our experiments, we found that protein ligation by protein  trans -splicing depended not only on the splicing junction sequences, but also on the foreign extein sequences. Furthermore, we could classify the protein  trans -splicing reactions in foreign contexts with a simple kinetic model into three groups according to their kinetic parameters in the presence of various reducing agents.    Conclusion  The shorter C-intein of the newly engineered split intein could be a useful tool for biotechnological applications including protein modification, incorporation of chemical probes, and segmental isotopic labelling. Based on kinetic analysis of the protein splicing reactions, we propose a general strategy to improve ligation yields by protein  trans -splicing, which could significantly enhance the applications of protein ligation by protein  trans -splicing.

In Vivo and In Vitro Protein Ligation by Naturally Occurring and Engineered Split DnaE Inteins