Activity-based selection of a proteolytic species using ribosome display

Area of Science:

• Biochemistry
• Molecular Biology
• Enzyme Engineering

Background:

• Ribosome display is a powerful in vitro protein evolution technique.
• Enzyme activity-based selection is crucial for functional protein isolation.
• Proteases are vital enzymes with diverse biological roles.

Purpose of the Study:

• To investigate the feasibility of displaying functional proteases using ribosome display.
• To establish a method for capturing active proteases based on their catalytic activity.
• To develop a screening platform for protease inhibitors and novel proteolytic enzymes.

Main Methods:

• Construction of ribosome display templates for bacterial sortase A (SrtA) using overlap PCR.
• Fusion of SrtA open reading frame with a flexible linker and an eGFP-derived tether.
• In vitro capture of SrtA-displaying complexes using an immobilized protease inhibitor (E-64) and subsequent mRNA recovery via RT-PCR.

Main Results:

• Functional expression and display of active SrtA protease in vitro were achieved.
• Specific capture of SrtA ternary complexes was demonstrated using catalytic activity-dependent binding.
• Capture was abolished with a catalytically inactive SrtA mutant and reduced by free inhibitor competition.

Conclusions:

• Ribosome display can be effectively utilized to display and select active proteases based on enzymatic function.
• This method provides a sensitive platform for screening protease inhibitors.
• The technique holds potential for the discovery and characterization of novel proteases.