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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 Smt3 and GB1 by the newly engineered split NpuDnaE intein.Time courses and kinetic analysis of protein ligation in the presence of (a) 0.5 mM TCEP or (b) 50 mM DTT. SDS-PAGE: 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, 24 hours.
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pone-0005185-g006: Protein ligation of Smt3 and GB1 by the newly engineered split NpuDnaE intein.Time courses and kinetic analysis of protein ligation in the presence of (a) 0.5 mM TCEP or (b) 50 mM DTT. SDS-PAGE: 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, 24 hours.

Mentions: Because of the strong influence of the extein sequences on protein trans-splicing, we wanted to test another small protein with a similar size, the yeast ubiquitin-like protein Smt3, for protein ligation [31]. Protein ligation of His-tagged Smt3 and GB1 by NpuDnaE-IntN123 was tested in the presence of either 50 mM DTT or 0.5 mM TCEP. The protein ligation of Smt3 and GB1 responded differently to the two different reducing agents. When 0.5 mM TCEP was used, the yield was more than 60–70%. On the contrary, only about 10% of the protein ligation was achieved in the presence of 50 mM DTT, where the cleavage reaction dominated. In this case the kinetic constant for trans-splicing in presence of 0.5 mM TCEP was estimated to be 8.3±0.7×10−5 (s−1). DTT induced the dominant cleavage reaction with a kinetic constant of 9.2±1.2×10−4 (s−1) (Figure 6). The protein ligation yield in the presence of 50 mM DTT is expected to be around 10% as it can be derived from Eq. (III) with an assumption that trans-splicing rates are similar for both DTT and TCEP. This is in good agreement with the yield obtained experimentally, suggesting that the simple model is appropriate for roughly estimating the yield without any intricate methods.


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 Smt3 and GB1 by the newly engineered split NpuDnaE intein.Time courses and kinetic analysis of protein ligation in the presence of (a) 0.5 mM TCEP or (b) 50 mM DTT. SDS-PAGE: 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, 24 hours.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664965&req=5

pone-0005185-g006: Protein ligation of Smt3 and GB1 by the newly engineered split NpuDnaE intein.Time courses and kinetic analysis of protein ligation in the presence of (a) 0.5 mM TCEP or (b) 50 mM DTT. SDS-PAGE: 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, 24 hours.
Mentions: Because of the strong influence of the extein sequences on protein trans-splicing, we wanted to test another small protein with a similar size, the yeast ubiquitin-like protein Smt3, for protein ligation [31]. Protein ligation of His-tagged Smt3 and GB1 by NpuDnaE-IntN123 was tested in the presence of either 50 mM DTT or 0.5 mM TCEP. The protein ligation of Smt3 and GB1 responded differently to the two different reducing agents. When 0.5 mM TCEP was used, the yield was more than 60–70%. On the contrary, only about 10% of the protein ligation was achieved in the presence of 50 mM DTT, where the cleavage reaction dominated. In this case the kinetic constant for trans-splicing in presence of 0.5 mM TCEP was estimated to be 8.3±0.7×10−5 (s−1). DTT induced the dominant cleavage reaction with a kinetic constant of 9.2±1.2×10−4 (s−1) (Figure 6). The protein ligation yield in the presence of 50 mM DTT is expected to be around 10% as it can be derived from Eq. (III) with an assumption that trans-splicing rates are similar for both DTT and TCEP. This is in good agreement with the yield obtained experimentally, suggesting that the simple model is appropriate for roughly estimating the yield without any intricate methods.

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