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

Kanamycin resistance is dependent on intein splicing in KanR site C. Three inteins were tested to ensure that resistance to kanamycin required a functional intein. E. coli were transformed with plasmids carrying the KanR gene with either an active or a catalytically inactive intein in site C. Growth after overnight incubation was then tested on plates with 40 μg/ml Kan. A, shown is growth at 37 °C with cells transformed with plasmids carrying the native Npu DnaE intein, an inactive Npu DnaE intein (C1A, N138D, C+1A), or a no intein control. B, shown is growth at 30 °C with cells transformed with plasmids carrying the native Mtu RecA intein, an inactive Mtu RecA intein (C1A, N440D, C+1A), the native MP-Be DnaB intein, or an inactive MP-Be DnaB intein (C320S).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Kanamycin resistance is dependent on intein splicing in KanR site C. Three inteins were tested to ensure that resistance to kanamycin required a functional intein. E. coli were transformed with plasmids carrying the KanR gene with either an active or a catalytically inactive intein in site C. Growth after overnight incubation was then tested on plates with 40 μg/ml Kan. A, shown is growth at 37 °C with cells transformed with plasmids carrying the native Npu DnaE intein, an inactive Npu DnaE intein (C1A, N138D, C+1A), or a no intein control. B, shown is growth at 30 °C with cells transformed with plasmids carrying the native Mtu RecA intein, an inactive Mtu RecA intein (C1A, N440D, C+1A), the native MP-Be DnaB intein, or an inactive MP-Be DnaB intein (C320S).

Mentions: Inteins flanked by three native N- and C-terminal extein residues were cloned separately into site C or D and tested for the ability of splicing to restore KanR (Fig. 1). Catalytically inactivated inteins (see “Experimental Procedures”) were likewise inserted into the same sites to ensure that KanR required splicing. The inteins tested were the Mtu-H37Rv RecA intein flanked by N-terminal Lys-Asn-Lys and C-terminal Cys-Ser-Pro, the MP-Be DnaB intein flanked by N-terminal Gln-Asp-Gln and C-terminal Thr-Lys-Asn, and a cis construct of the naturally split Npu PCC73102 DnaE intein (16, 25) flanked by N-terminal Ala-Glu-Tyr and C-terminal Cys-Phe-Asn (Fig. 2 and data not shown). KanR was observed with the native inteins but not with the inactive inteins. These results validate the use of these two sites for assessing intein functionality. However, site C showed far greater tolerance for variable amino acid incorporation than site D and would thus make the best system for high throughput analysis of extein sequences.


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)

Kanamycin resistance is dependent on intein splicing in KanR site C. Three inteins were tested to ensure that resistance to kanamycin required a functional intein. E. coli were transformed with plasmids carrying the KanR gene with either an active or a catalytically inactive intein in site C. Growth after overnight incubation was then tested on plates with 40 μg/ml Kan. A, shown is growth at 37 °C with cells transformed with plasmids carrying the native Npu DnaE intein, an inactive Npu DnaE intein (C1A, N138D, C+1A), or a no intein control. B, shown is growth at 30 °C with cells transformed with plasmids carrying the native Mtu RecA intein, an inactive Mtu RecA intein (C1A, N440D, C+1A), the native MP-Be DnaB intein, or an inactive MP-Be DnaB intein (C320S).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Kanamycin resistance is dependent on intein splicing in KanR site C. Three inteins were tested to ensure that resistance to kanamycin required a functional intein. E. coli were transformed with plasmids carrying the KanR gene with either an active or a catalytically inactive intein in site C. Growth after overnight incubation was then tested on plates with 40 μg/ml Kan. A, shown is growth at 37 °C with cells transformed with plasmids carrying the native Npu DnaE intein, an inactive Npu DnaE intein (C1A, N138D, C+1A), or a no intein control. B, shown is growth at 30 °C with cells transformed with plasmids carrying the native Mtu RecA intein, an inactive Mtu RecA intein (C1A, N440D, C+1A), the native MP-Be DnaB intein, or an inactive MP-Be DnaB intein (C320S).
Mentions: Inteins flanked by three native N- and C-terminal extein residues were cloned separately into site C or D and tested for the ability of splicing to restore KanR (Fig. 1). Catalytically inactivated inteins (see “Experimental Procedures”) were likewise inserted into the same sites to ensure that KanR required splicing. The inteins tested were the Mtu-H37Rv RecA intein flanked by N-terminal Lys-Asn-Lys and C-terminal Cys-Ser-Pro, the MP-Be DnaB intein flanked by N-terminal Gln-Asp-Gln and C-terminal Thr-Lys-Asn, and a cis construct of the naturally split Npu PCC73102 DnaE intein (16, 25) flanked by N-terminal Ala-Glu-Tyr and C-terminal Cys-Phe-Asn (Fig. 2 and data not shown). KanR was observed with the native inteins but not with the inactive inteins. These results validate the use of these two sites for assessing intein functionality. However, site C showed far greater tolerance for variable amino acid incorporation than site D and would thus make the best system for high throughput analysis of extein sequences.

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