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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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In vitro protein ligation of nSH3 and GB1 by the naturally occurring split NpuDnaE intein.(a) Time course of the protein ligation of nSH3 and GB1 by naturally occurring split NpuDnaE intein in the presence of 50 mM DTT. 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. (c) SDS-PAGE analysis of the ligation reaction after overnight incubation in the presence of different reducing agents.
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pone-0005185-g004: In vitro protein ligation of nSH3 and GB1 by the naturally occurring split NpuDnaE intein.(a) Time course of the protein ligation of nSH3 and GB1 by naturally occurring split NpuDnaE intein in the presence of 50 mM DTT. 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. (c) SDS-PAGE analysis of the ligation reaction after overnight incubation in the presence of different reducing agents.

Mentions: The robustness of naturally split NpuDnaE intein encouraged us to use NpuDnaE intein as a general tool for protein ligation and to apply it to biologically relevant proteins [24]. The Src homology 3 (SH3) domain is one of the most abundant domains in multi-domain proteins. Therefore, we were interested in protein ligation of the two SH3 domains from c-Crk-II adaptor protein [27]. Despite the robustness of NpuDnaE intein, protein ligation of the two SH3 domains by wild-type NpuDnaE intein was not possible, because the side reactions were dominating the trans-splicing and producing mainly cleaved products (Figure 3a and 3c). When the N-terminal SH3 (nSH3) was replaced with the model protein GB1, both in vivo and in vitro ligation of the two proteins by protein trans-splicing was still not possible with high yields (Figure 3b and 3d, Figure S4). On the other hand, the ligation of the two proteins in vitro as well as in vivo was significantly improved after replacing the C-terminal SH3 (cSH3) with GB1 (Figure 4a, Table 1). These observations indicate that protein trans-splicing can be significantly influenced not only by the sequences near the splicing junctions but also by the exteins, which brings additional complexity to protein trans-splicing. Furthermore, the replacement of the C-terminal precursor protein suggests that the C-terminal fragment containing cSH3 negatively affects the protein ligation.


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)

In vitro protein ligation of nSH3 and GB1 by the naturally occurring split NpuDnaE intein.(a) Time course of the protein ligation of nSH3 and GB1 by naturally occurring split NpuDnaE intein in the presence of 50 mM DTT. 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. (c) SDS-PAGE analysis of the ligation reaction after overnight incubation in the presence of different reducing agents.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005185-g004: In vitro protein ligation of nSH3 and GB1 by the naturally occurring split NpuDnaE intein.(a) Time course of the protein ligation of nSH3 and GB1 by naturally occurring split NpuDnaE intein in the presence of 50 mM DTT. 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. (c) SDS-PAGE analysis of the ligation reaction after overnight incubation in the presence of different reducing agents.
Mentions: The robustness of naturally split NpuDnaE intein encouraged us to use NpuDnaE intein as a general tool for protein ligation and to apply it to biologically relevant proteins [24]. The Src homology 3 (SH3) domain is one of the most abundant domains in multi-domain proteins. Therefore, we were interested in protein ligation of the two SH3 domains from c-Crk-II adaptor protein [27]. Despite the robustness of NpuDnaE intein, protein ligation of the two SH3 domains by wild-type NpuDnaE intein was not possible, because the side reactions were dominating the trans-splicing and producing mainly cleaved products (Figure 3a and 3c). When the N-terminal SH3 (nSH3) was replaced with the model protein GB1, both in vivo and in vitro ligation of the two proteins by protein trans-splicing was still not possible with high yields (Figure 3b and 3d, Figure S4). On the other hand, the ligation of the two proteins in vitro as well as in vivo was significantly improved after replacing the C-terminal SH3 (cSH3) with GB1 (Figure 4a, Table 1). These observations indicate that protein trans-splicing can be significantly influenced not only by the sequences near the splicing junctions but also by the exteins, which brings additional complexity to protein trans-splicing. Furthermore, the replacement of the C-terminal precursor protein suggests that the C-terminal fragment containing cSH3 negatively affects the protein ligation.

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