Faster Protein Splicing with the Nostoc punctiforme DnaE Intein Using Non-native Extein Residues

Francine Perler,Manoj Cheriyan,Chandra Sekhar Pedamallu,Kazuo Tori

doi:10.1074/jbc.m112.433094

Francine Perler, Manoj Cheriyan + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m112.433094

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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.

Highlights

Inteins are protein maturation machines that enable technologies for regulating enzymes and protein semisynthesis
Because of the ease of use and high stringency of antibiotic-based selections, we focused our efforts on identifying sites within C. diphtheriae aminoglycoside phosphotransferase (Aph), an enzyme responsible for kanamycin resistance (KanR)
Using Phusion polymerase-based inverted PCR, we created 10 libraries where either (a) six codons at a chosen site were replaced with six consecutive in-frame NNK codons or (b) six in-frame NNK codons were inserted into the middle of the chosen sequence, where N represents any nucleotide and K represents G or C. These libraries represent the scar that would be left in the KanR protein after splicing when three flanking amino acids are included at each end of the intein

Summary

Introduction

Inteins are protein maturation machines that enable technologies for regulating enzymes and protein semisynthesis. We developed a genetic selection for splicingdependent 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

Results

Discussion

Conclusion