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

A. Sesilja Aranko*, Sara Züger, Edith Buchinger, Hideo Iwaï

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

59 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article numbere5185
Pages (from-to)1-10
JournalPloS one
Volume4
Issue number4
DOIs
Publication statusPublished - 13 Apr 2009
MoE publication typeA1 Journal article-refereed

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