Two-strain, cell-selective protein labeling in mixed bacterial cultures

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Cell-selective metabolic labeling is crucial for studying specific cell subpopulations in complex biological systems.
• Existing tools, like azidonorleucine (Anl) activated by NLL-MetRS, enable labeling but expanding the available amino acids is necessary.

Purpose of the Study:

• To develop a new methionyl-tRNA synthetase (MetRS) variant capable of activating propargylglycine (Pra) for cell-selective protein labeling.
• To enable differential labeling of proteins from distinct cell populations within a mixed culture.

Main Methods:

• Directed evolution, including active-site randomization and error-prone PCR, was used to engineer a novel MetRS variant (PraRS).
• PraRS was tested for its ability to incorporate propargylglycine (Pra) into proteins in a Met-auxotrophic Escherichia coli strain.
• In-gel fluorescence was used to confirm Pra incorporation after conjugation with Cy5-azide.
• Differential labeling was demonstrated in a co-culture of two bacterial strains using both NLL-MetRS and PraRS.

Main Results:

• A MetRS variant, PraRS, was successfully engineered to efficiently incorporate propargylglycine (Pra) into proteins.
• Proteins produced by E. coli expressing PraRS were labeled with Pra, confirmed by fluorescence.
• Combined use of NLL-MetRS and PraRS enabled distinct, cell-selective labeling of proteins from two co-cultured bacterial strains.
• Sequential click chemistry reactions allowed for the unambiguous identification of the cellular origin of each labeled protein.

Conclusions:

• The engineered PraRS enzyme expands the toolkit for cell-selective metabolic labeling with noncanonical amino acids.
• This method allows for differential protein labeling in mixed cell populations, facilitating the study of complex biological interactions.
• The developed system provides a robust approach for distinguishing protein origins in co-cultured systems.