A Structure-Based Assembly Screen of Protein Cage Libraries in Living Cells: Experimentally Repacking a...

Area of Science:

• Biochemistry
• Nanotechnology
• Synthetic Biology

Background:

• Protein cages are hollow nanoscale capsules with diverse applications in drug delivery, nanomaterials, and catalysis.
• They serve as model systems for studying protein-protein interactions in cellular nanostructure formation.
• Protein cages are increasingly utilized as scaffolds in synthetic biology.

Purpose of the Study:

• To adapt a fluorescence-based protein cage assembly assay for use in living cells.
• To develop a high-throughput screening method using flow cytometry for protein cage libraries.
• To demonstrate the assay's utility by screening engineered mini-ferritin variants.

Main Methods:

• Expansion of a fluorescence-based protein cage assembly assay to living cells.
• Implementation of a high-throughput screening platform utilizing flow cytometry.
• Application of the assay to screen libraries of a double-alanine mutant of DNA-binding protein from starved cells (Dps).

Main Results:

• Successful adaptation of the protein cage assembly assay for high-throughput screening in living cells.
• Identification of engineered mini-ferritin (Dps) variants with recovered cage formation after randomization of residues around a disruptive double mutation.
• Demonstration of the assay's effectiveness in identifying functional protein cage assemblies from randomized libraries.

Conclusions:

• The developed flow cytometry assay is a powerful tool for high-throughput screening of protein cage libraries in living cells.
• This approach enables the engineering of protein cages with tailored properties and functionalities.
• The study validates the utility of the assay for directed evolution of protein cage assembly.