HaloTag display enables quantitative single-particle characterisation and functionalisation of engineered...

Area of Science:

• Biotechnology
• Nanomedicine
• Cell Biology

Background:

• Extracellular vesicles (EVs) are crucial for intercellular communication and are engineered for therapeutics.
• Expressing proteins on EV surfaces enhances targeting and bioactivity.
• Quantifying engineered proteins on single EVs is challenging but vital for characterization.

Purpose of the Study:

• To develop and validate a HaloTag-based platform for precise quantification of engineered proteins on EVs.
• To compare HaloTag-labeling with traditional antibody-labeling methods.
• To demonstrate the utility of HaloTag for optimizing EV bioengineering strategies.

Main Methods:

• Development of a HaloTag-based system for covalent and stoichiometric attachment of labels to engineered EV surface proteins.
• Evaluation using orthogonal methods like flow cytometry and fluorescence microscopy.
• Comparison of HaloTag-labeling with antibody-labeling using single-vesicle flow cytometry.

Main Results:

• HaloTag-mediated quantification of surface proteins on EVs is robust across different analysis methods.
• Antibody-labeling significantly underestimates protein incorporation compared to HaloTag-labeling.
• The HaloTag system effectively facilitates comparison of different protein designs for EV bioengineering.

Conclusions:

• The HaloTag system provides a reliable and accurate method for single-EV protein quantification.
• This platform overcomes limitations of existing methods, particularly antibody-based approaches.
• HaloTag is a valuable tool for advancing EV bioengineering and therapeutic development.