Genetic Code Expansion Method for Temporal Labeling of Endogenously Expressed Proteins

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Accurate spatiotemporal labeling of endogenous proteins is crucial for understanding cellular dynamics.
• Existing methods may lack precision or interfere with physiological processes.

Purpose of the Study:

• To develop and validate a method for high-resolution, spatiotemporal labeling of endogenous proteins.
• To enable quantification of protein synthesis rates and localization at the single-cell level.

Main Methods:

• Utilized genetic code expansion with an orthogonal tRNA/tRNA synthetase pair.
• Employed CRISPR/Cas9 genome engineering for precise protein labeling.
• Incorporated noncanonical amino acids for stop codon read-through events.
• Applied proximity ligation assay and real-time imaging for analysis.

Main Results:

• Successfully pulse-labeled endogenous β-actin and tumor protein p53 with an HA tag.
• Demonstrated seamless quantification of protein synthesis rates and spatial localization.
• Confirmed no interference with physiological cellular expression control.
• Observed no perturbation of endogenous protein functions.

Conclusions:

• The combined genetic code expansion and CRISPR/Cas9 method provides a powerful tool for studying endogenous protein dynamics.
• This approach offers high spatiotemporal resolution and minimal disruption to cellular processes.
• Enables precise quantification of protein synthesis and localization in real-time.