TAT hitchhiker selection expanded to folding helpers, multimeric interactions and combinations with protein fragment...

Area of Science:

• Molecular Biology
• Biochemistry
• Protein Engineering

Background:

• The twin-arginine translocation (TAT) pathway selectively transports folded proteins across the bacterial membrane.
• This unique feature can be exploited for novel protein selection and engineering strategies.

Purpose of the Study:

• To develop and demonstrate modular selection systems based on TAT-dependent translocation for directed evolution.
• To engineer proteins that enhance folding, mediate protein-protein interactions, and identify disease-relevant peptides.

Main Methods:

• Utilized TAT pathway for selection systems, including direct translocation selection and hitchhiker translocation (HiT) selection.
• Combined HiT with protein fragment complementation for heterotrimeric interactions.
• Engineered TEM β-lactamase fragments for enhanced activity and stability.

Main Results:

• Successfully selected folding enhancers and proteins mediating dimeric, oligomeric, and heterotrimeric interactions.
• Demonstrated correlation between cellular fitness and biophysical protein properties.
• Identified high-affinity peptides targeting leukemia and melanoma proteins (MITF and ETO) via coiled-coil or tetra-helix-bundle formation.

Conclusions:

• The developed TAT selection systems are versatile tools for directed evolution and protein engineering.
• These methods enable the discovery of peptides that inhibit disease-promoting interactions, addressing previously challenging selection problems.