Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Inteins are protein sequences that catalyze their own excision and ligation of flanking protein sequences (exteins).
• Intein chemistry enables biotechnological applications like enzyme control and peptide ligation.
• Studying intein substrate specificity is challenging due to the vast sequence possibilities.

Purpose of the Study:

• To develop a method to systematically study intein substrate specificity.
• To identify novel extein sequences that facilitate intein splicing.
• To assess the generalizability and kinetic efficiency of newly identified intein substrates.

Main Methods:

• A genetic selection system was created based on kanamycin resistance, dependent on protein splicing.
• Randomized flanking sequences (six amino acids) were tested for their ability to support splicing of the Nostoc punctiforme Npu DnaE intein.
• Kinetic analysis was performed on selected extein sequences.

Main Results:

• The study identified a much wider range of extein sequences that support Npu DnaE intein splicing than previously known.
• Little specificity was observed for N-extein sequences, while two C-extein positions were found to be critical.
• Novel extein sequences promoted splicing in unrelated proteins and exhibited splicing rates comparable to or faster than native exteins.

Conclusions:

• The Npu DnaE intein exhibits broader substrate specificity than previously recognized.
• The findings provide new, generalizable intein insertion sites for protein engineering.
• Naturally occurring flanking sequences are not necessarily optimal for intein splicing efficiency.