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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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Logo diagrams of flanking extein sequences for 483 randomly chosen positive hits selected for splicing of the Npu DnaE intein in KanR site C were made using the Weblogo program (28). The vertical red line indicates the position of the intein. The +1 position was fixed as Cys. A, shown is the entire library of 483 clones. B, shown are all clones with Trp+2. C, shown are all clones with Met+2. D, shown are all clones with Gly at −3 or −2. E, shown are all clones with Trp+2 and a hydrophilic residue at +3. F, shown is all clones with Trp+2 and a hydrophobic residue at +3.
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Figure 3: Logo diagrams of flanking extein sequences for 483 randomly chosen positive hits selected for splicing of the Npu DnaE intein in KanR site C were made using the Weblogo program (28). The vertical red line indicates the position of the intein. The +1 position was fixed as Cys. A, shown is the entire library of 483 clones. B, shown are all clones with Trp+2. C, shown are all clones with Met+2. D, shown are all clones with Gly at −3 or −2. E, shown are all clones with Trp+2 and a hydrophilic residue at +3. F, shown is all clones with Trp+2 and a hydrophobic residue at +3.

Mentions: Because it is not practical to individually sequence all of the selected clones, the entire Aph and Npu DnaE intein precursor gene was sequenced from a representative, randomly chosen set of clones. Using an automated program we verified that 483 of the selected plasmids contained unambiguous sequences with no mutations in the Aph and the Npu DnaE intein genes. Examination of these extein sequences showed evidence of selection at some positions but not others. The largest specificity preferences were observed at the +2 and +3 positions of the C- extein (Table 1 and Fig. 3A). The natural splice site (Phe+2 and Asn+3) was not observed in the 483 sequenced clones. Instead, 92% of the selected sequences possessed Trp+2, and 7% contained Met+2 (Table 1 and Fig. 3). Phe, Val, Cys, and Lys were observed once, and Tyr was observed twice at the +2 position. The non-selected library confirmed that all residues were present at the +2 position in the expected quantities (data not shown), but Trp was selected over the naturally occurring Phe+2. The preference at the +3 position was for Tyr and residues similar to the natural Asn+3, with Asn+3, Asp+3, Gln+3, and Glu+3 representing 49% of the total hits (Table 1). The range of N-terminal extein sequences that allow splicing was very broad; however, it was evidently important to maintain flexibility at the N terminus because the selected hits have an abundance of Gly at the −3 and −2 positions (Table 1 and Fig. 3D).


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 diagrams of flanking extein sequences for 483 randomly chosen positive hits selected for splicing of the Npu DnaE intein in KanR site C were made using the Weblogo program (28). The vertical red line indicates the position of the intein. The +1 position was fixed as Cys. A, shown is the entire library of 483 clones. B, shown are all clones with Trp+2. C, shown are all clones with Met+2. D, shown are all clones with Gly at −3 or −2. E, shown are all clones with Trp+2 and a hydrophilic residue at +3. F, shown is all clones with Trp+2 and a hydrophobic residue at +3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Logo diagrams of flanking extein sequences for 483 randomly chosen positive hits selected for splicing of the Npu DnaE intein in KanR site C were made using the Weblogo program (28). The vertical red line indicates the position of the intein. The +1 position was fixed as Cys. A, shown is the entire library of 483 clones. B, shown are all clones with Trp+2. C, shown are all clones with Met+2. D, shown are all clones with Gly at −3 or −2. E, shown are all clones with Trp+2 and a hydrophilic residue at +3. F, shown is all clones with Trp+2 and a hydrophobic residue at +3.
Mentions: Because it is not practical to individually sequence all of the selected clones, the entire Aph and Npu DnaE intein precursor gene was sequenced from a representative, randomly chosen set of clones. Using an automated program we verified that 483 of the selected plasmids contained unambiguous sequences with no mutations in the Aph and the Npu DnaE intein genes. Examination of these extein sequences showed evidence of selection at some positions but not others. The largest specificity preferences were observed at the +2 and +3 positions of the C- extein (Table 1 and Fig. 3A). The natural splice site (Phe+2 and Asn+3) was not observed in the 483 sequenced clones. Instead, 92% of the selected sequences possessed Trp+2, and 7% contained Met+2 (Table 1 and Fig. 3). Phe, Val, Cys, and Lys were observed once, and Tyr was observed twice at the +2 position. The non-selected library confirmed that all residues were present at the +2 position in the expected quantities (data not shown), but Trp was selected over the naturally occurring Phe+2. The preference at the +3 position was for Tyr and residues similar to the natural Asn+3, with Asn+3, Asp+3, Gln+3, and Glu+3 representing 49% of the total hits (Table 1). The range of N-terminal extein sequences that allow splicing was very broad; however, it was evidently important to maintain flexibility at the N terminus because the selected hits have an abundance of Gly at the −3 and −2 positions (Table 1 and Fig. 3D).

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