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

Aranko AS, Züger S, Buchinger E, Iwaï H - PLoS ONE (2009)

Bottom Line: PCC6803 and from Nostoc punctiforme.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.

View Article: PubMed Central - PubMed

Affiliation: Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

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.

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Protein ligation of two SH3 domains by the newly engineered split NpuDnaE intein.(a) SDS-PAGE analysis of the time course from the protein ligation reaction of nSH3 and cSH3 in the presence of 0.5 mM TCEP. Lane 1, 0 min after the mixing; lane 2, 3 min; lane 3, 10 min; lane 4, 30 min; lane 5, 1 hour; lane 6, 3 hours; lane 7, 22 hours. (b) Kinetic analysis of the protein ligation from the SDS-PAGE.
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pone-0005185-g005: Protein ligation of two SH3 domains by the newly engineered split NpuDnaE intein.(a) SDS-PAGE analysis of the time course from the protein ligation reaction of nSH3 and cSH3 in the presence of 0.5 mM TCEP. Lane 1, 0 min after the mixing; lane 2, 3 min; lane 3, 10 min; lane 4, 30 min; lane 5, 1 hour; lane 6, 3 hours; lane 7, 22 hours. (b) Kinetic analysis of the protein ligation from the SDS-PAGE.

Mentions: From the aforementioned results with the SH3 domains, we assumed that the C-intein fused with cSH3 is the limiting factor for the protein ligation of two SH3 domains, inducing fast cleavage reactions. We believe that cSH3 probably interferes with association of N- and C-inteins of wild-type NpuDnaE intein and that the shorter C-intein might not interfere the ligation of the two SH3 domains. Therefore, we decided to replace the intein with the newly engineered NpuDnaE intein (NpuDnaE-IntN123/NpuDnaE-IntC15) for the ligation. As demonstrated in Figure 5, the new split NpuDnaE intein could indeed ligate nSH3 and cSH3 that were not possible to be ligated by the naturally occurring split NpuDnaE intein. It demonstrates the effectiveness of the shorter C-intein in the case of difficult ligations such as the one between the two SH3 domains. The kinetic constants for trans-splicing were estimated to be 4.8±0.3×10−5 (s−1) in the presence of 0.5 mM TCEP (Figure 5b). Thus, the engineered split NpuDnaE intein can significantly improve the protein ligation by accelerating the trans-splicing reaction.


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

Aranko AS, Züger S, Buchinger E, Iwaï H - PLoS ONE (2009)

Protein ligation of two SH3 domains by the newly engineered split NpuDnaE intein.(a) SDS-PAGE analysis of the time course from the protein ligation reaction of nSH3 and cSH3 in the presence of 0.5 mM TCEP. Lane 1, 0 min after the mixing; lane 2, 3 min; lane 3, 10 min; lane 4, 30 min; lane 5, 1 hour; lane 6, 3 hours; lane 7, 22 hours. (b) Kinetic analysis of the protein ligation from the SDS-PAGE.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664965&req=5

pone-0005185-g005: Protein ligation of two SH3 domains by the newly engineered split NpuDnaE intein.(a) SDS-PAGE analysis of the time course from the protein ligation reaction of nSH3 and cSH3 in the presence of 0.5 mM TCEP. Lane 1, 0 min after the mixing; lane 2, 3 min; lane 3, 10 min; lane 4, 30 min; lane 5, 1 hour; lane 6, 3 hours; lane 7, 22 hours. (b) Kinetic analysis of the protein ligation from the SDS-PAGE.
Mentions: From the aforementioned results with the SH3 domains, we assumed that the C-intein fused with cSH3 is the limiting factor for the protein ligation of two SH3 domains, inducing fast cleavage reactions. We believe that cSH3 probably interferes with association of N- and C-inteins of wild-type NpuDnaE intein and that the shorter C-intein might not interfere the ligation of the two SH3 domains. Therefore, we decided to replace the intein with the newly engineered NpuDnaE intein (NpuDnaE-IntN123/NpuDnaE-IntC15) for the ligation. As demonstrated in Figure 5, the new split NpuDnaE intein could indeed ligate nSH3 and cSH3 that were not possible to be ligated by the naturally occurring split NpuDnaE intein. It demonstrates the effectiveness of the shorter C-intein in the case of difficult ligations such as the one between the two SH3 domains. The kinetic constants for trans-splicing were estimated to be 4.8±0.3×10−5 (s−1) in the presence of 0.5 mM TCEP (Figure 5b). Thus, the engineered split NpuDnaE intein can significantly improve the protein ligation by accelerating the trans-splicing reaction.

Bottom Line: PCC6803 and from Nostoc punctiforme.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.

View Article: PubMed Central - PubMed

Affiliation: Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

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.

Show MeSH