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
Splicing of the Npu DnaE intein with the extein sequences listed in Table 2 fused to either KanR or luciferase.A, shown are KanR-Npu DnaE intein fusions E1–E8, the native extein (NE), or KanR with an N-terminal His tag but no intein (Kan). B, shown are KanR-Npu DnaE intein fusions E10–E20. Lane L contains the NEB prestained broad range (10–230 kDa) Ladder. Western blots A and B were probed with Mouse IgG anti-His tag antibody followed by detection with LI-COR IRDye 680 goat anti-mouse secondary antibody. C, shown are luciferase-Npu DnaE intein fusions with selected or native (NE) flanking extein sequences. Lane D, a mutated, catalytically inactive intein fusion is shown. The asterisk band is the result of proteolysis of the precursor in the inactive intein sample. Western blot C was probed with Mouse IgG anti-firefly luciferase antibody followed by detection with LI-COR IRDye 680 goat anti-mouse secondary antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Splicing of the Npu DnaE intein with the extein sequences listed in Table 2 fused to either KanR or luciferase.A, shown are KanR-Npu DnaE intein fusions E1–E8, the native extein (NE), or KanR with an N-terminal His tag but no intein (Kan). B, shown are KanR-Npu DnaE intein fusions E10–E20. Lane L contains the NEB prestained broad range (10–230 kDa) Ladder. Western blots A and B were probed with Mouse IgG anti-His tag antibody followed by detection with LI-COR IRDye 680 goat anti-mouse secondary antibody. C, shown are luciferase-Npu DnaE intein fusions with selected or native (NE) flanking extein sequences. Lane D, a mutated, catalytically inactive intein fusion is shown. The asterisk band is the result of proteolysis of the precursor in the inactive intein sample. Western blot C was probed with Mouse IgG anti-firefly luciferase antibody followed by detection with LI-COR IRDye 680 goat anti-mouse secondary antibody.

Mentions: Twenty positive hits representative of the range of sequence diversity found at the N- and C-terminal exteins were chosen for further analysis (Table 2). Cells containing these plasmids were grown in LB media plus Amp overnight, and Western blotting was used to directly confirm whether splicing occurred. Antibody probing based on the presence of an N-terminal His tag was used to detect precursor and the mature KanR product. All hits tested displayed robust in vivo splicing (∼95%) with no detectable amount of cleavage products (Fig. 4 and data not shown).


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)

Splicing of the Npu DnaE intein with the extein sequences listed in Table 2 fused to either KanR or luciferase.A, shown are KanR-Npu DnaE intein fusions E1–E8, the native extein (NE), or KanR with an N-terminal His tag but no intein (Kan). B, shown are KanR-Npu DnaE intein fusions E10–E20. Lane L contains the NEB prestained broad range (10–230 kDa) Ladder. Western blots A and B were probed with Mouse IgG anti-His tag antibody followed by detection with LI-COR IRDye 680 goat anti-mouse secondary antibody. C, shown are luciferase-Npu DnaE intein fusions with selected or native (NE) flanking extein sequences. Lane D, a mutated, catalytically inactive intein fusion is shown. The asterisk band is the result of proteolysis of the precursor in the inactive intein sample. Western blot C was probed with Mouse IgG anti-firefly luciferase antibody followed by detection with LI-COR IRDye 680 goat anti-mouse secondary antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Splicing of the Npu DnaE intein with the extein sequences listed in Table 2 fused to either KanR or luciferase.A, shown are KanR-Npu DnaE intein fusions E1–E8, the native extein (NE), or KanR with an N-terminal His tag but no intein (Kan). B, shown are KanR-Npu DnaE intein fusions E10–E20. Lane L contains the NEB prestained broad range (10–230 kDa) Ladder. Western blots A and B were probed with Mouse IgG anti-His tag antibody followed by detection with LI-COR IRDye 680 goat anti-mouse secondary antibody. C, shown are luciferase-Npu DnaE intein fusions with selected or native (NE) flanking extein sequences. Lane D, a mutated, catalytically inactive intein fusion is shown. The asterisk band is the result of proteolysis of the precursor in the inactive intein sample. Western blot C was probed with Mouse IgG anti-firefly luciferase antibody followed by detection with LI-COR IRDye 680 goat anti-mouse secondary antibody.
Mentions: Twenty positive hits representative of the range of sequence diversity found at the N- and C-terminal exteins were chosen for further analysis (Table 2). Cells containing these plasmids were grown in LB media plus Amp overnight, and Western blotting was used to directly confirm whether splicing occurred. Antibody probing based on the presence of an N-terminal His tag was used to detect precursor and the mature KanR product. All hits tested displayed robust in vivo splicing (∼95%) with no detectable amount of cleavage products (Fig. 4 and data not shown).

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