Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Protein trans-splicing utilizes split inteins to ligate separate protein fragments.
• The dnaE gene encodes a crucial subunit of DNA polymerase III.

Purpose of the Study:

• To investigate the protein trans-splicing activity of the naturally split intein from Nostoc punctiforme dnaE (Npu DnaE) in Escherichia coli.
• To compare the efficiency and characteristics of Npu DnaE with the Ssp DnaE intein.

Main Methods:

• Expression of split Npu DnaE intein fragments and non-native exteins in Escherichia coli.
• Assessing trans-splicing efficiency through protein analysis.
• Evaluating the impact of domain swapping between Npu DnaE and Ssp DnaE.
• Testing the tolerance of Npu DnaE to amino acid substitutions in extein sequences.

Main Results:

• Npu DnaE demonstrated robust trans-splicing activity with >98% efficiency in E. coli, surpassing Ssp DnaE.
• N- and C-terminal fragments of Npu DnaE could be interchanged with Ssp DnaE fragments without compromising activity.
• Npu DnaE exhibited enhanced tolerance to amino acid variations in the C-terminal extein.

Conclusions:

• Npu DnaE is a highly efficient and versatile split intein for protein trans-splicing applications.
• Its robustness and flexibility make it a valuable tool in protein engineering and synthetic biology.
• Npu DnaE offers advantages over Ssp DnaE for certain protein ligation strategies.