A Novel "Prebinding" Strategy Dramatically Enhances Sortase-Mediated Coupling of Proteins to Liposomes

Area of Science:

• Biochemistry
• Biotechnology
• Materials Science

Background:

• Sortase A enzymes mediate protein ligation but face limitations in efficiency and yield.
• Protein-liposome coupling is crucial for various biotechnological applications.
• Existing methods are often slow and hindered by enzyme hydrolysis.

Purpose of the Study:

• To quantitatively assess sortase A-mediated protein-liposome coupling efficiency and kinetics.
• To overcome limitations of slow coupling rates and low yields.
• To develop an improved method for creating protein-modified liposomes.

Main Methods:

• Utilized model substrate proteins (GFP, SNAP-tag) and liposomes with acceptor lipids.
• Investigated sortase A (S. aureus, S. pyogenes) activity under standard conditions.
• Developed and tested a 'prebinding' strategy using oligohistidine tags and Ni(II)-chelating lipids.

Main Results:

• Standard sortase A coupling is slow, yields are modest, and limited by enzyme hydrolysis.
• The prebinding approach significantly enhances coupling rates and yields.
• Low sortase concentrations achieved high efficiency with minimized hydrolysis using the prebinding method.

Conclusions:

• The prebinding strategy effectively overcomes limitations in sortase A-mediated protein-liposome coupling.
• This method offers high efficiency, yield, and adaptability for diverse applications.
• The approach can be rendered 'traceless' for various end uses of modified liposomes.