In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins

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.

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.

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.