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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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KanR selection construct used to study intein specificity for flanking extein sequences. KanR was used as a reporter for protein splicing with the Npu DnaE intein and three additional extein residues flanking both its N and C termini. The sequence of the native extein is shown inserted into KanR site C. Underlined amino acids were simultaneously randomized. In the numbering scheme used in this paper, residues in the KanR protein, the variable extein positions, and the intein were numbered independently as indicated. Positions in the KanR protein were numbered as in the original protein, ignoring insertions.
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Figure 1: KanR selection construct used to study intein specificity for flanking extein sequences. KanR was used as a reporter for protein splicing with the Npu DnaE intein and three additional extein residues flanking both its N and C termini. The sequence of the native extein is shown inserted into KanR site C. Underlined amino acids were simultaneously randomized. In the numbering scheme used in this paper, residues in the KanR protein, the variable extein positions, and the intein were numbered independently as indicated. Positions in the KanR protein were numbered as in the original protein, ignoring insertions.

Mentions: Hand-mixed primers (forward, 5′-CT GAT TCC GGA GAA NNK NNK NNK TGC CTG AGC TAT GA; reverse, 5′-A CAC TGC TAG CGC ATC AAC AAT ATT MNN MNN GCA ATT AGA GGC AAT AA) were ordered from Integrated DNA Technologies (Coralville, IA) to introduce three variable codons flanking the intein N terminus and two variable codons flanking the intein C terminus after a fixed extein Cys residue (Fig. 1). In these primers N = A, T, G, or C, K = G or T, and M = C or A. Primers also included restriction enzyme sites for cloning into site C in the kanamycin resistance gene and sequences that matched the template, which was the cis construct of the Npu DnaE intein (16). PCR products using these primers were cloned into plasmids that contained mutations at the natural Aph ribosome binding site (RBS) changing GGGGTGTTATG to GAAGGTGTTCATG, which reduces the level of expression by ∼20-fold.5


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)

KanR selection construct used to study intein specificity for flanking extein sequences. KanR was used as a reporter for protein splicing with the Npu DnaE intein and three additional extein residues flanking both its N and C termini. The sequence of the native extein is shown inserted into KanR site C. Underlined amino acids were simultaneously randomized. In the numbering scheme used in this paper, residues in the KanR protein, the variable extein positions, and the intein were numbered independently as indicated. Positions in the KanR protein were numbered as in the original protein, ignoring insertions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: KanR selection construct used to study intein specificity for flanking extein sequences. KanR was used as a reporter for protein splicing with the Npu DnaE intein and three additional extein residues flanking both its N and C termini. The sequence of the native extein is shown inserted into KanR site C. Underlined amino acids were simultaneously randomized. In the numbering scheme used in this paper, residues in the KanR protein, the variable extein positions, and the intein were numbered independently as indicated. Positions in the KanR protein were numbered as in the original protein, ignoring insertions.
Mentions: Hand-mixed primers (forward, 5′-CT GAT TCC GGA GAA NNK NNK NNK TGC CTG AGC TAT GA; reverse, 5′-A CAC TGC TAG CGC ATC AAC AAT ATT MNN MNN GCA ATT AGA GGC AAT AA) were ordered from Integrated DNA Technologies (Coralville, IA) to introduce three variable codons flanking the intein N terminus and two variable codons flanking the intein C terminus after a fixed extein Cys residue (Fig. 1). In these primers N = A, T, G, or C, K = G or T, and M = C or A. Primers also included restriction enzyme sites for cloning into site C in the kanamycin resistance gene and sequences that matched the template, which was the cis construct of the Npu DnaE intein (16). PCR products using these primers were cloned into plasmids that contained mutations at the natural Aph ribosome binding site (RBS) changing GGGGTGTTATG to GAAGGTGTTCATG, which reduces the level of expression by ∼20-fold.5

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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