SPLICEFINDER - a fast and easy screening method for active protein trans-splicing positions

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Protein trans-splicing (PTS) enables ligation of protein fragments for modification and function control.
• The flanking amino acids at splice junctions significantly impact PTS activity.
• Predicting split intein activity in foreign exteins remains challenging.

Purpose of the Study:

• To develop a method for screening active split intein insertions in target proteins.
• To enable reliable prediction of split intein activity in diverse protein contexts.
• To facilitate applications of protein trans-splicing.

Main Methods:

• Development of SPLICEFINDER, a PCR-based screening method.
• Application of SPLICEFINDER for screening split intein activity in various exteins.
• Demonstration of SPLICEFINDER for segmental isotopic labeling and multi-domain protein generation.

Main Results:

• SPLICEFINDER allows fast and easy screening of active split intein insertions.
• The method is applicable to any target protein.
• Successful demonstration of segmental isotopic labeling and generation of multi-domain, enzymatically active proteins.

Conclusions:

• SPLICEFINDER is a valuable tool for protein engineering and functional studies.
• The method overcomes limitations in predicting split intein activity.
• Facilitates advanced protein modification and construction applications.