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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 vivo protein ligations by the newly engineered split SspDnaE and NpuDnaE inteins.(a) SDS-PAGE analysis of in vivo protein ligations by the newly engineered split SspDnaE inteins after purification with Ni-NTA. The combinations of SspDnaE-IntN and SspDnaE-IntC are indicated on the top of the lanes. (b) In vivo protein ligation by NpuDnaE intein with the newly engineered split site (NpuDnaE-IntN123/C15). Lane 1, before induction; lane 2, 1.5 hours after induction only with arabinose; lane 3, 1.5 hours after additional induction with IPTG; lane 4, 3 hours after induction with IPTG and arabinose; lane 5, elution from Ni-NTA column.
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pone-0005185-g002: In vivo protein ligations by the newly engineered split SspDnaE and NpuDnaE inteins.(a) SDS-PAGE analysis of in vivo protein ligations by the newly engineered split SspDnaE inteins after purification with Ni-NTA. The combinations of SspDnaE-IntN and SspDnaE-IntC are indicated on the top of the lanes. (b) In vivo protein ligation by NpuDnaE intein with the newly engineered split site (NpuDnaE-IntN123/C15). Lane 1, before induction; lane 2, 1.5 hours after induction only with arabinose; lane 3, 1.5 hours after additional induction with IPTG; lane 4, 3 hours after induction with IPTG and arabinose; lane 5, elution from Ni-NTA column.

Mentions: Protein ligation by the new split inteins was tested in vivo using the dual vector system previously developed in our group [8]. This system allows us to conveniently check protein ligation because protein ligation could be initiated by the induction of the two precursor fragments with the two inducers, isopropyl-β-D-thiogalactoside (IPTG) and arabinose, and subsequently analyzed by SDS-PAGE [24]. Moreover, endogenous auxiliary factors such as chaperones might improve protein ligation in cells by promoting correct protein folding. The C-terminal part was always first induced for 0.5 hours ensuring an excess of the C-terminal precursor prior to the expression of the N-terminal precursor, and followed by the induction of the N-terminal precursor for another 3.5–5.5 hours. The pre-existing C-terminal precursor protein could be converted into the ligated product through protein trans-splicing after the association with the N-terminal part and protein splicing. The expression level of the N-terminal fragment was monitored by SDS-PAGE in order to avoid an enormous excess of the N-terminal part, which could underestimate the ligation yields. Immobilized Metal Affinity Chromatography (IMAC) was used to purify the N-terminal His-tagged precursor, the ligated product, and, if any, the cleaved N-terminal GB1 produced by the side reactions (Figure 1a). If in vivo protein ligation works with 100% efficiency and if there is no excess of the N-terminal precursor, only H6-GB1-CBD will be purified by IMAC through the N-terminal His-tag. If the N- and C-terminal fragments associate with each other but no protein splicing is induced, both N- and C-terminal fragments (H6-GB1-SspDnaE-IntN and SspDnaE-IntC-CBD) will be purified owing to the affinity between them. Furthermore, if the N- and C-inteins do not interact or if the C-terminal cleavage reaction is the dominant reaction after association of the N- and C-inteins, a single band of the N-terminal precursor is expected to be visible in the SDS gel. In some cases, during protein purification and sample preparation for SDS-PAGE, reactions such as splicing and cleavages could take place, which produced smaller bands of cleaved and spliced products. The ligated product was confirmed by mass-spectrometry (Figure S2). We could identify the ligated product H6-GB1-CBD in the elution fractions from IMAC only for the combinations of SspDnaE-IntN123/SspDnaE-IntC36 (wild-type), SspDnaE-IntN130/SspDnaE-IntC30, SspDnaE-IntN137/SspDnaE-IntC23, and SspDnaE-IntN144/SspDnaE-IntC16 (Figure 2a). The ligation yields were estimated from the ratios between the intensities of the ligated product and one of the most abundant residual precursor fragments in the SDS gel, which were ca. 3% for SspDnaE-IntN144/SspDnaE-IntC16, ca. 1% for SspDnaE-IntN137/SspDnaE-IntC23, and ca. 16% for SspDnaE-IntN130/SspDnaE-IntC30. These efficiencies might be underestimated if an excess of the N-terminal part was present during the expression due to the co-purification of the N-terminal precursor containing an N-terminal His-tag. The highest yield was estimated for the wild-type combination of SspDnaE-IntN123/SspDnaE-IntC36 (67%). Albeit the amounts of the ligated products produced by the newly engineered inteins were very small, the protein ligation was still detectable by SDS-PAGE. The split site of SspDnaE-IntN144/SspDnaE-IntC16 was the split site of the shortest C-intein retaining detectable splicing activity. However, the ligation efficiency was significantly lower than that of wild-type SspDnaE intein because of the low splicing activity and the side reactions. The pairs of SspDnaE-IntN151/SspDnaE-IntC9 and SspDnaE-IntN154/SspDnaE-IntC6 could not induce protein trans-splicing as only the N-terminal precursor was purified, indicating there was no significant interaction between them. On the other hand, the shortest C-intein construct of SspDnaE-IntC3 was purified together with the N-terminal SspDnaE-IntN157 indicating that there was sufficient interaction between them. However, we could not identify any ligated product although there was a band at 18.4 kDa in the SDS gel indicating a small amount of the N-cleavage reaction that produced IntN.


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 vivo protein ligations by the newly engineered split SspDnaE and NpuDnaE inteins.(a) SDS-PAGE analysis of in vivo protein ligations by the newly engineered split SspDnaE inteins after purification with Ni-NTA. The combinations of SspDnaE-IntN and SspDnaE-IntC are indicated on the top of the lanes. (b) In vivo protein ligation by NpuDnaE intein with the newly engineered split site (NpuDnaE-IntN123/C15). Lane 1, before induction; lane 2, 1.5 hours after induction only with arabinose; lane 3, 1.5 hours after additional induction with IPTG; lane 4, 3 hours after induction with IPTG and arabinose; lane 5, elution from Ni-NTA column.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664965&req=5

pone-0005185-g002: In vivo protein ligations by the newly engineered split SspDnaE and NpuDnaE inteins.(a) SDS-PAGE analysis of in vivo protein ligations by the newly engineered split SspDnaE inteins after purification with Ni-NTA. The combinations of SspDnaE-IntN and SspDnaE-IntC are indicated on the top of the lanes. (b) In vivo protein ligation by NpuDnaE intein with the newly engineered split site (NpuDnaE-IntN123/C15). Lane 1, before induction; lane 2, 1.5 hours after induction only with arabinose; lane 3, 1.5 hours after additional induction with IPTG; lane 4, 3 hours after induction with IPTG and arabinose; lane 5, elution from Ni-NTA column.
Mentions: Protein ligation by the new split inteins was tested in vivo using the dual vector system previously developed in our group [8]. This system allows us to conveniently check protein ligation because protein ligation could be initiated by the induction of the two precursor fragments with the two inducers, isopropyl-β-D-thiogalactoside (IPTG) and arabinose, and subsequently analyzed by SDS-PAGE [24]. Moreover, endogenous auxiliary factors such as chaperones might improve protein ligation in cells by promoting correct protein folding. The C-terminal part was always first induced for 0.5 hours ensuring an excess of the C-terminal precursor prior to the expression of the N-terminal precursor, and followed by the induction of the N-terminal precursor for another 3.5–5.5 hours. The pre-existing C-terminal precursor protein could be converted into the ligated product through protein trans-splicing after the association with the N-terminal part and protein splicing. The expression level of the N-terminal fragment was monitored by SDS-PAGE in order to avoid an enormous excess of the N-terminal part, which could underestimate the ligation yields. Immobilized Metal Affinity Chromatography (IMAC) was used to purify the N-terminal His-tagged precursor, the ligated product, and, if any, the cleaved N-terminal GB1 produced by the side reactions (Figure 1a). If in vivo protein ligation works with 100% efficiency and if there is no excess of the N-terminal precursor, only H6-GB1-CBD will be purified by IMAC through the N-terminal His-tag. If the N- and C-terminal fragments associate with each other but no protein splicing is induced, both N- and C-terminal fragments (H6-GB1-SspDnaE-IntN and SspDnaE-IntC-CBD) will be purified owing to the affinity between them. Furthermore, if the N- and C-inteins do not interact or if the C-terminal cleavage reaction is the dominant reaction after association of the N- and C-inteins, a single band of the N-terminal precursor is expected to be visible in the SDS gel. In some cases, during protein purification and sample preparation for SDS-PAGE, reactions such as splicing and cleavages could take place, which produced smaller bands of cleaved and spliced products. The ligated product was confirmed by mass-spectrometry (Figure S2). We could identify the ligated product H6-GB1-CBD in the elution fractions from IMAC only for the combinations of SspDnaE-IntN123/SspDnaE-IntC36 (wild-type), SspDnaE-IntN130/SspDnaE-IntC30, SspDnaE-IntN137/SspDnaE-IntC23, and SspDnaE-IntN144/SspDnaE-IntC16 (Figure 2a). The ligation yields were estimated from the ratios between the intensities of the ligated product and one of the most abundant residual precursor fragments in the SDS gel, which were ca. 3% for SspDnaE-IntN144/SspDnaE-IntC16, ca. 1% for SspDnaE-IntN137/SspDnaE-IntC23, and ca. 16% for SspDnaE-IntN130/SspDnaE-IntC30. These efficiencies might be underestimated if an excess of the N-terminal part was present during the expression due to the co-purification of the N-terminal precursor containing an N-terminal His-tag. The highest yield was estimated for the wild-type combination of SspDnaE-IntN123/SspDnaE-IntC36 (67%). Albeit the amounts of the ligated products produced by the newly engineered inteins were very small, the protein ligation was still detectable by SDS-PAGE. The split site of SspDnaE-IntN144/SspDnaE-IntC16 was the split site of the shortest C-intein retaining detectable splicing activity. However, the ligation efficiency was significantly lower than that of wild-type SspDnaE intein because of the low splicing activity and the side reactions. The pairs of SspDnaE-IntN151/SspDnaE-IntC9 and SspDnaE-IntN154/SspDnaE-IntC6 could not induce protein trans-splicing as only the N-terminal precursor was purified, indicating there was no significant interaction between them. On the other hand, the shortest C-intein construct of SspDnaE-IntC3 was purified together with the N-terminal SspDnaE-IntN157 indicating that there was sufficient interaction between them. However, we could not identify any ligated product although there was a band at 18.4 kDa in the SDS gel indicating a small amount of the N-cleavage reaction that produced IntN.

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