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Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Published on: February 10, 2023

Debugging Eukaryotic Genetic Code Expansion for Site-Specific Click-PAINT Super-Resolution Microscopy.

Ivana Nikić^1,2, Gemma Estrada Girona¹, Jun Hee Kang¹

¹Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117, Heidelberg, Germany.

Angewandte Chemie (International Ed. in English)

|November 3, 2016

Summary

This summary is machine-generated.

Genetic code expansion enables precise protein labeling for super-resolution microscopy. This improved system enhances efficiency and reduces background noise, advancing protein engineering in eukaryotes.

Keywords:

PAINT click chemistry genetic code expansion protein labeling super-resolution microscopy

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Area of Science:

Biochemistry
Molecular Biology
Microscopy

Background:

Super-resolution microscopy (SRM) requires small, photostable fluorescent labels for proteins.
Genetic code expansion (GCE) allows incorporating noncanonical amino acids (ncAAs) for labeling.
Current GCE methods suffer from low ncAA incorporation and high background fluorescence, limiting SRM utility.

Purpose of the Study:

To enhance the efficiency and reduce background noise of GCE for SRM applications.
To enable visualization of low-abundance proteins in mammalian cells using GCE and SRM.
To improve protein engineering in eukaryotes via a reengineered GCE system.

Main Methods:

Redirecting the subcellular localization of a pyrrolysine-based GCE system.
Utilizing click chemistry for labeling with ncAAs.
Employing DNA-PAINT microscopy for high-resolution imaging.

Main Results:

Achieved residue-specific protein labeling with high precision.
Eliminated untargeted background fluorescence.
Substantially boosted protein expression yield.
Enabled visualization of low-abundance proteins in mammalian cells.

Conclusions:

The reengineered GCE system significantly improves protein labeling for SRM.
This approach offers a versatile, biocompatible, and potentially unbleachable labeling method.
The enhanced GCE system has broad implications for protein engineering in eukaryotes.