In vitro selection for catalytic activity with ribosome display

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Ribosome display is a cell-free protein evolution technique.
• Enzyme evolution often relies on cellular systems, limiting sequence space exploration.
• Selecting for catalytic activity directly is challenging.

Purpose of the Study:

• To develop and demonstrate an in vitro selection method for catalytic activity using ribosome display.
• To validate the efficiency of ribosome display for selecting enzymes based on turnover.
• To compare selection for catalytic activity with selection for binding affinity.

Main Methods:

• Functional display of RTEM-beta-lactamase on ribosomes with its encoding mRNA.
• Design and synthesis of a mechanism-based inhibitor (biotinylated ampicillin sulfone) for beta-lactamase.
• Enrichment of active enzyme through multiple rounds of ribosome display selection.
• Comparison with selection for binding using beta-lactamase inhibitory protein (BLIP).

Main Results:

• Successful functional display of RTEM-beta-lactamase.
• Demonstrated enrichment of active enzyme over 100-fold per selection cycle.
• Ribosome display showed comparable efficiency for catalytic activity and affinity selections.
• Validated the use of a suicide inhibitor for in vitro catalytic selection.

Conclusions:

• Ribosome display enables direct in vitro selection for catalytic activity.
• This cell-free approach significantly expands the explorable sequence space for enzyme engineering.
• The method holds promise for discovering novel enzymes with desired catalytic functions.