A transferrin receptor-based vector enables robust Type-II membrane protein display on mammalian cells

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Cell surface protein display is crucial for protein engineering in mammalian systems.
• Existing display systems primarily support type-I membrane proteins, limiting applications for type-II proteins.
• Type-II membrane proteins have an inverted topology, posing challenges for current display technologies.

Purpose of the Study:

• To develop a novel and generalizable surface display system for type-II membrane proteins in mammalian cells.
• To enable the study and engineering of type-II membrane proteins using cell surface display.
• To overcome the topological limitations of existing display platforms.

Main Methods:

• Development of a transferrin receptor 1 (TfR1)-based display vector.
• Leveraging the native TfR1 cytoplasmic and transmembrane domains to enforce type-II topology.
• Fusion of a target type-II membrane protein (TMEM106b) to the TfR1 scaffold.
• Validation using fluorescence-activated cell sorting (FACS) and confocal microscopy.

Main Results:

• The TfR1-based vector successfully directed type-II membrane protein display to the cell surface.
• Robust cell-surface localization of the model type-II protein TMEM106b was achieved.
• The system demonstrated efficient trafficking to the plasma membrane, confirming correct topology enforcement.

Conclusions:

• A novel TfR1-based system provides a robust platform for type-II membrane protein display in mammalian cells.
• This system expands the utility of cell surface display for a broader range of membrane proteins.
• The developed vector offers a generalizable tool for protein engineering and functional studies of type-II proteins.