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A Conserved Histidine Residue Drives Extein Dependence in an Enhanced Atypically Split Intein
Giridhar Sekar1, Adam J Stevens2, Anahita Z Mostafavi2
1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States.
Journal of the American Chemical Society
|October 4, 2022
Summary
Researchers engineered a new protein trans-splicing (PTS) system, Cat*, to overcome limitations caused by flanking extein sequences. This enhanced system shows improved activity with challenging N-extein residues, expanding PTS applications.
Area of Science:
- Biotechnology
- Chemical Biology
- Molecular Biology
Background:
- Split intein-mediated protein trans-splicing (PTS) is crucial for protein ligation in biotechnology.
- Extein sequences flanking inteins can impede splicing efficiency, limiting PTS applications.
- Atypical split inteins, like 'Cat', show high sensitivity to N-extein sequences.
Purpose of the Study:
- To engineer an improved split intein system with enhanced tolerance to unfavorable N-extein residues.
- To investigate the mechanistic basis of extein dependence in atypical split inteins.
- To expand the utility of split intein technology in protein engineering.
Main Methods:
- Directed evolution using error-prone PCR and cell-based selection to generate enhanced inteins.
- Solution nuclear magnetic resonance (NMR) spectroscopy to study protein dynamics.
- Molecular dynamics (MD) simulations to analyze residue interactions and conformational changes.
Main Results:
- Developed 'Cat*', an engineered split intein exhibiting significantly enhanced PTS activity with problematic N-extein sequences.
- Identified the critical role of a conserved B-block histidine residue (His78) dynamics in mediating extein dependence.
- Demonstrated that Cat* overcomes previously observed extein-related splicing limitations.
Conclusions:
- The engineered Cat* intein broadens the applicability of atypically split inteins in protein ligation.
- Understanding the dynamics of key residues like His78 provides insights into extein-intein interactions.
- This work advances the development of robust protein engineering tools for biotechnology.
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