Protein Chemical Modification Inside Living Cells Using Split Inteins

Area of Science:

• Cell biology
• Biochemistry
• Molecular biology

Background:

• Visualizing and manipulating proteins in cells is crucial for understanding biological processes.
• Fluorescent proteins are useful but have limitations due to interdependent spectral and structural properties.
• Chemical labeling offers an alternative for protein studies in vivo.

Purpose of the Study:

• To develop and demonstrate a novel method for site-specific protein labeling and fluorescence activation in living cells.
• To overcome the limitations of traditional fluorescent protein-based methods.
• To enable control over protein function and localization.

Main Methods:

• Utilized fluorescence resonance energy transfer (FRET)-quenched DnaE split-inteins for protein labeling.
• Applied the method for site-specific labeling of the DNA binding domain (DBD) of transcription factor YY1.
• Tested the approach in various human cell lines.

Main Results:

• Successfully achieved site-specific labeling and fluorescence activation of proteins in living cells.
• Demonstrated the labeling of the YY1 transcription factor's DBD.
• Showed potential for modifying proteins to control cellular localization and biological activity.

Conclusions:

• The FRET-quenched DnaE split-intein system provides a powerful tool for site-specific protein manipulation in cells.
• This method overcomes limitations of fluorescent proteins, enabling new possibilities in cell biology research.
• The approach can be used to modulate protein function and localization, advancing studies of cellular processes.