Limits...
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.

Show MeSH

Related in: MedlinePlus

Logo diagram of flanking extein sequences identified in the low stringency pilot study with the native KanR RBS reveals expanded specificity at the +2 and +3 positions. This figure was made using the Weblogo program (28) and shows 38 randomly chosen positive hits selected for splicing of the Npu DnaE intein in KanR site C. This construct results in expression of more KanR protein than needed for resistance and, therefore, allows selection of precursors that splice slowly or with a low percentage of spliced product (data not shown). The vertical red line indicates the position of the intein. The +1 position was fixed as Cys.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3585056&req=5

Figure 5: Logo diagram of flanking extein sequences identified in the low stringency pilot study with the native KanR RBS reveals expanded specificity at the +2 and +3 positions. This figure was made using the Weblogo program (28) and shows 38 randomly chosen positive hits selected for splicing of the Npu DnaE intein in KanR site C. This construct results in expression of more KanR protein than needed for resistance and, therefore, allows selection of precursors that splice slowly or with a low percentage of spliced product (data not shown). The vertical red line indicates the position of the intein. The +1 position was fixed as Cys.

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)

Logo diagram of flanking extein sequences identified in the low stringency pilot study with the native KanR RBS reveals expanded specificity at the +2 and +3 positions. This figure was made using the Weblogo program (28) and shows 38 randomly chosen positive hits selected for splicing of the Npu DnaE intein in KanR site C. This construct results in expression of more KanR protein than needed for resistance and, therefore, allows selection of precursors that splice slowly or with a low percentage of spliced product (data not shown). The vertical red line indicates the position of the intein. The +1 position was fixed as Cys.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585056&req=5

Figure 5: Logo diagram of flanking extein sequences identified in the low stringency pilot study with the native KanR RBS reveals expanded specificity at the +2 and +3 positions. This figure was made using the Weblogo program (28) and shows 38 randomly chosen positive hits selected for splicing of the Npu DnaE intein in KanR site C. This construct results in expression of more KanR protein than needed for resistance and, therefore, allows selection of precursors that splice slowly or with a low percentage of spliced product (data not shown). The vertical red line indicates the position of the intein. The +1 position was fixed as Cys.
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