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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 trans-splicing and locations of the new split sites.(a) Schematic representation of the protein trans-splicing process and two possible side reactions of N- and C-cleavage. Two fragments of the protein of interest (POI) can be ligated by protein trans-splicing reaction. (b) Sequence alignment of SspDnaE and NpuDnaE inteins. The locations of the experimentally tested split sites of SspDnaE and NpuDnaE inteins are indicated by inverse triangles on the top of the primary sequences. The asterisks above inverse triangles indicate the naturally occurring split site. Filled triangles indicate the split sites, where the split inteins retained protein trans-splicing activity. Open triangles indicate the split sites, where no protein trans-splicing activity could be detected. The location of the b-strands observed in the crystal structures of SspDnaE intein (PDB code 1ZDE) [35] and SspDnaB mini-intein (1MI8) [36] are indicated at the bottom of the sequences. The numbering for b-strands is adapted from SspDnaB mini-intein [36].
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pone-0005185-g001: Protein trans-splicing and locations of the new split sites.(a) Schematic representation of the protein trans-splicing process and two possible side reactions of N- and C-cleavage. Two fragments of the protein of interest (POI) can be ligated by protein trans-splicing reaction. (b) Sequence alignment of SspDnaE and NpuDnaE inteins. The locations of the experimentally tested split sites of SspDnaE and NpuDnaE inteins are indicated by inverse triangles on the top of the primary sequences. The asterisks above inverse triangles indicate the naturally occurring split site. Filled triangles indicate the split sites, where the split inteins retained protein trans-splicing activity. Open triangles indicate the split sites, where no protein trans-splicing activity could be detected. The location of the b-strands observed in the crystal structures of SspDnaE intein (PDB code 1ZDE) [35] and SspDnaB mini-intein (1MI8) [36] are indicated at the bottom of the sequences. The numbering for b-strands is adapted from SspDnaB mini-intein [36].

Mentions: SspDnaE intein is one of the naturally occurring split inteins widely used in biotechnological applications. Naturally occurring split inteins can spontaneously induce protein splicing in trans after association of the N- and C-terminal parts (Figure 1a). In contrast, artificially split inteins often require tedious denaturation and renaturation steps to restore protein splicing activity because of lower solubility of the precursor fragments [23]. Protein ligation of two flanking foreign sequences through protein trans-splicing by naturally split inteins usually requires no additional cofactor, but a few residues of the original extein sequences might be necessary for efficient splicing [11]. To identify new functional split inteins with a shorter C-intein, we have moved the split site in naturally split SspDnaE intein towards the C-terminus by shortening the C-terminal half (SspDnaE-IntC) systematically by 6–7 residues and elongating the N-terminal half (SspDnaE-IntN) by approximately the same lengths (Figure 1b, Figure S1). SspDnaE-IntNs were fused with the N-terminally His-tagged B1 domain of protein G (GB1), of which expression was under the control of an inducible T7 promoter [8]. Previously, we found that the change of the N-terminal junction sequence of EY from SspDnaE to other sequences such as GS had little influence on the ligation yield [24]. Therefore, we used a linker of GS originated from the restriction site of BamHI between GB1 and SspDnaE-IntNs. SspDnaE-IntCs were also fused to a chitin binding domain (CBD), of which expression was controlled by an arabinose promoter (Figure S1). We kept the sequence of CFNK from the wild-type junction sequence of SspDnaE and added GT for the cloning site of KpnI as a linker between SspDnaE-IntCs and CBD [8]. GB1 and CBD were used here as model proteins because they are small soluble proteins. The N- and C-precursor proteins were genetically encoded into two separate plasmids that bear the compatible RSF3010 and ColE1 origins [8]. Seven plasmids for each half were constructed for testing in vivo and in vitro protein ligation (Table S1 and Figure S1).


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 trans-splicing and locations of the new split sites.(a) Schematic representation of the protein trans-splicing process and two possible side reactions of N- and C-cleavage. Two fragments of the protein of interest (POI) can be ligated by protein trans-splicing reaction. (b) Sequence alignment of SspDnaE and NpuDnaE inteins. The locations of the experimentally tested split sites of SspDnaE and NpuDnaE inteins are indicated by inverse triangles on the top of the primary sequences. The asterisks above inverse triangles indicate the naturally occurring split site. Filled triangles indicate the split sites, where the split inteins retained protein trans-splicing activity. Open triangles indicate the split sites, where no protein trans-splicing activity could be detected. The location of the b-strands observed in the crystal structures of SspDnaE intein (PDB code 1ZDE) [35] and SspDnaB mini-intein (1MI8) [36] are indicated at the bottom of the sequences. The numbering for b-strands is adapted from SspDnaB mini-intein [36].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664965&req=5

pone-0005185-g001: Protein trans-splicing and locations of the new split sites.(a) Schematic representation of the protein trans-splicing process and two possible side reactions of N- and C-cleavage. Two fragments of the protein of interest (POI) can be ligated by protein trans-splicing reaction. (b) Sequence alignment of SspDnaE and NpuDnaE inteins. The locations of the experimentally tested split sites of SspDnaE and NpuDnaE inteins are indicated by inverse triangles on the top of the primary sequences. The asterisks above inverse triangles indicate the naturally occurring split site. Filled triangles indicate the split sites, where the split inteins retained protein trans-splicing activity. Open triangles indicate the split sites, where no protein trans-splicing activity could be detected. The location of the b-strands observed in the crystal structures of SspDnaE intein (PDB code 1ZDE) [35] and SspDnaB mini-intein (1MI8) [36] are indicated at the bottom of the sequences. The numbering for b-strands is adapted from SspDnaB mini-intein [36].
Mentions: SspDnaE intein is one of the naturally occurring split inteins widely used in biotechnological applications. Naturally occurring split inteins can spontaneously induce protein splicing in trans after association of the N- and C-terminal parts (Figure 1a). In contrast, artificially split inteins often require tedious denaturation and renaturation steps to restore protein splicing activity because of lower solubility of the precursor fragments [23]. Protein ligation of two flanking foreign sequences through protein trans-splicing by naturally split inteins usually requires no additional cofactor, but a few residues of the original extein sequences might be necessary for efficient splicing [11]. To identify new functional split inteins with a shorter C-intein, we have moved the split site in naturally split SspDnaE intein towards the C-terminus by shortening the C-terminal half (SspDnaE-IntC) systematically by 6–7 residues and elongating the N-terminal half (SspDnaE-IntN) by approximately the same lengths (Figure 1b, Figure S1). SspDnaE-IntNs were fused with the N-terminally His-tagged B1 domain of protein G (GB1), of which expression was under the control of an inducible T7 promoter [8]. Previously, we found that the change of the N-terminal junction sequence of EY from SspDnaE to other sequences such as GS had little influence on the ligation yield [24]. Therefore, we used a linker of GS originated from the restriction site of BamHI between GB1 and SspDnaE-IntNs. SspDnaE-IntCs were also fused to a chitin binding domain (CBD), of which expression was controlled by an arabinose promoter (Figure S1). We kept the sequence of CFNK from the wild-type junction sequence of SspDnaE and added GT for the cloning site of KpnI as a linker between SspDnaE-IntCs and CBD [8]. GB1 and CBD were used here as model proteins because they are small soluble proteins. The N- and C-precursor proteins were genetically encoded into two separate plasmids that bear the compatible RSF3010 and ColE1 origins [8]. Seven plasmids for each half were constructed for testing in vivo and in vitro protein ligation (Table S1 and Figure S1).

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