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Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues.

Cheriyan M, Pedamallu CS, Tori K, Perler F - J. Biol. Chem. (2013)

Bottom Line: We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions.The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target.Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, Inc, Ipswich, Massachusetts 01938, USA.

ABSTRACT
Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.

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Kinetic analysis of trans-splicing inteins with selected flanking extein sequences.A, shown is a schematic representation of the trans-splicing reaction. The N-terminal precursor fragment (M-IN) consists of the maltose binding protein (M or MBP) followed by a short linker, three native or selected extein residues, and the N-terminal half of the Npu DnaE intein (IN: residues 1–102). The C-terminal precursor fragment (IC-PHis) consists of the C-terminal half of the Npu DnaE intein (IC; residues 103–138), three native or selected extein residues, a short linker, the ΔSal fragment of paramyosin (P) and a C-terminal His tag. M-IN and IC-PHis spontaneously assemble when mixed resulting in intein activation and protein splicing. Any unreacted precursor complex and the IN-IC product complex dissociate during SDS-PAGE sample preparation. B, Western blots show the time course of trans-splicing with native (Ala-Glu-Tyr/Cys-Phe-Asn, left panel) or E6 (Arg-Gly-Lys/Cys-Trp-Glu, right panel) flanking extein residues. Time points from 0 to 5 min are listed across the top of each blot. The nitrocellulose membranes were simultaneously probed with mouse IgG anti-His tag antibody and rabbit anti-MBP antiserum followed by detection with LI-COR IRDye 680 goat anti-mouse (red) and IRDye 800 goat anti-rabbit secondary antibodies (green). The band labeled M-PHis was quantified to measure the rate of spliced product formation. The asterisk marks endogenous E. coli MBP. Lane Lad contains the NEB prestained broad range (10–230 kDa) ladder. C, shown are plots of the percent conversion of precursor to product during the trans-splicing time courses with native (left panel) or E6 (right panel) flanking extein residues. The red line represents the fit of the data to a first order decay reaction, and the S.D. is indicated by the error bars.
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Figure 6: Kinetic analysis of trans-splicing inteins with selected flanking extein sequences.A, shown is a schematic representation of the trans-splicing reaction. The N-terminal precursor fragment (M-IN) consists of the maltose binding protein (M or MBP) followed by a short linker, three native or selected extein residues, and the N-terminal half of the Npu DnaE intein (IN: residues 1–102). The C-terminal precursor fragment (IC-PHis) consists of the C-terminal half of the Npu DnaE intein (IC; residues 103–138), three native or selected extein residues, a short linker, the ΔSal fragment of paramyosin (P) and a C-terminal His tag. M-IN and IC-PHis spontaneously assemble when mixed resulting in intein activation and protein splicing. Any unreacted precursor complex and the IN-IC product complex dissociate during SDS-PAGE sample preparation. B, Western blots show the time course of trans-splicing with native (Ala-Glu-Tyr/Cys-Phe-Asn, left panel) or E6 (Arg-Gly-Lys/Cys-Trp-Glu, right panel) flanking extein residues. Time points from 0 to 5 min are listed across the top of each blot. The nitrocellulose membranes were simultaneously probed with mouse IgG anti-His tag antibody and rabbit anti-MBP antiserum followed by detection with LI-COR IRDye 680 goat anti-mouse (red) and IRDye 800 goat anti-rabbit secondary antibodies (green). The band labeled M-PHis was quantified to measure the rate of spliced product formation. The asterisk marks endogenous E. coli MBP. Lane Lad contains the NEB prestained broad range (10–230 kDa) ladder. C, shown are plots of the percent conversion of precursor to product during the trans-splicing time courses with native (left panel) or E6 (right panel) flanking extein residues. The red line represents the fit of the data to a first order decay reaction, and the S.D. is indicated by the error bars.

Mentions: To further validate the selected extein sequences, we examined the kinetics of trans-splicing of the naturally split Npu DnaE intein using either the native flanking extein residues or sequences that represent commonly selected motifs (Table 3). As controls, an inactive Npu DnaE intein mutant and a variant identified in the low stringency pilot selection (EP) were also tested (Fig. 5). M-IN and IC-PHis fragments (Fig. 6A) were expressed separately in NEB Express cells by induction with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside at 25 °C for 4 h. Cells were harvested, and the kinetics of trans-splicing were assayed at 30 °C over a 10-min time course using the soluble fraction of the lysate. Samples taken at various time points were electrophoresed on SDS-PAGE followed by Western blot analysis to quantify spliced products (Fig. 6B). In the homologous Ssp DnaE intein, association of the IN and IC fragments was shown to approach diffusion-controlled limits (26, 27). Assuming that the Npu DnaE intein IN and IC fragments associate in a similarly rapid manor, the kinetics of trans-splicing can be fit to a first order decay reaction when the concentration of M-IN is maintained at excess over the concentration of IC-PHis.


Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues.

Cheriyan M, Pedamallu CS, Tori K, Perler F - J. Biol. Chem. (2013)

Kinetic analysis of trans-splicing inteins with selected flanking extein sequences.A, shown is a schematic representation of the trans-splicing reaction. The N-terminal precursor fragment (M-IN) consists of the maltose binding protein (M or MBP) followed by a short linker, three native or selected extein residues, and the N-terminal half of the Npu DnaE intein (IN: residues 1–102). The C-terminal precursor fragment (IC-PHis) consists of the C-terminal half of the Npu DnaE intein (IC; residues 103–138), three native or selected extein residues, a short linker, the ΔSal fragment of paramyosin (P) and a C-terminal His tag. M-IN and IC-PHis spontaneously assemble when mixed resulting in intein activation and protein splicing. Any unreacted precursor complex and the IN-IC product complex dissociate during SDS-PAGE sample preparation. B, Western blots show the time course of trans-splicing with native (Ala-Glu-Tyr/Cys-Phe-Asn, left panel) or E6 (Arg-Gly-Lys/Cys-Trp-Glu, right panel) flanking extein residues. Time points from 0 to 5 min are listed across the top of each blot. The nitrocellulose membranes were simultaneously probed with mouse IgG anti-His tag antibody and rabbit anti-MBP antiserum followed by detection with LI-COR IRDye 680 goat anti-mouse (red) and IRDye 800 goat anti-rabbit secondary antibodies (green). The band labeled M-PHis was quantified to measure the rate of spliced product formation. The asterisk marks endogenous E. coli MBP. Lane Lad contains the NEB prestained broad range (10–230 kDa) ladder. C, shown are plots of the percent conversion of precursor to product during the trans-splicing time courses with native (left panel) or E6 (right panel) flanking extein residues. The red line represents the fit of the data to a first order decay reaction, and the S.D. is indicated by the error bars.
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Figure 6: Kinetic analysis of trans-splicing inteins with selected flanking extein sequences.A, shown is a schematic representation of the trans-splicing reaction. The N-terminal precursor fragment (M-IN) consists of the maltose binding protein (M or MBP) followed by a short linker, three native or selected extein residues, and the N-terminal half of the Npu DnaE intein (IN: residues 1–102). The C-terminal precursor fragment (IC-PHis) consists of the C-terminal half of the Npu DnaE intein (IC; residues 103–138), three native or selected extein residues, a short linker, the ΔSal fragment of paramyosin (P) and a C-terminal His tag. M-IN and IC-PHis spontaneously assemble when mixed resulting in intein activation and protein splicing. Any unreacted precursor complex and the IN-IC product complex dissociate during SDS-PAGE sample preparation. B, Western blots show the time course of trans-splicing with native (Ala-Glu-Tyr/Cys-Phe-Asn, left panel) or E6 (Arg-Gly-Lys/Cys-Trp-Glu, right panel) flanking extein residues. Time points from 0 to 5 min are listed across the top of each blot. The nitrocellulose membranes were simultaneously probed with mouse IgG anti-His tag antibody and rabbit anti-MBP antiserum followed by detection with LI-COR IRDye 680 goat anti-mouse (red) and IRDye 800 goat anti-rabbit secondary antibodies (green). The band labeled M-PHis was quantified to measure the rate of spliced product formation. The asterisk marks endogenous E. coli MBP. Lane Lad contains the NEB prestained broad range (10–230 kDa) ladder. C, shown are plots of the percent conversion of precursor to product during the trans-splicing time courses with native (left panel) or E6 (right panel) flanking extein residues. The red line represents the fit of the data to a first order decay reaction, and the S.D. is indicated by the error bars.
Mentions: To further validate the selected extein sequences, we examined the kinetics of trans-splicing of the naturally split Npu DnaE intein using either the native flanking extein residues or sequences that represent commonly selected motifs (Table 3). As controls, an inactive Npu DnaE intein mutant and a variant identified in the low stringency pilot selection (EP) were also tested (Fig. 5). M-IN and IC-PHis fragments (Fig. 6A) were expressed separately in NEB Express cells by induction with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside at 25 °C for 4 h. Cells were harvested, and the kinetics of trans-splicing were assayed at 30 °C over a 10-min time course using the soluble fraction of the lysate. Samples taken at various time points were electrophoresed on SDS-PAGE followed by Western blot analysis to quantify spliced products (Fig. 6B). In the homologous Ssp DnaE intein, association of the IN and IC fragments was shown to approach diffusion-controlled limits (26, 27). Assuming that the Npu DnaE intein IN and IC fragments associate in a similarly rapid manor, the kinetics of trans-splicing can be fit to a first order decay reaction when the concentration of M-IN is maintained at excess over the concentration of IC-PHis.

Bottom Line: We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions.The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target.Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, Inc, Ipswich, Massachusetts 01938, USA.

ABSTRACT
Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.

Show MeSH
Related in: MedlinePlus