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An in vitro compartmentalization-based method for the selection of bond-forming enzymes from large libraries
Paul Gianella1, Erik L Snapp2, Matthew Levy3,4
1Department of Biochemistry, Albert Einstein College of Medicine, 1301 Morris Park Ave, Bronx, New York, 10461.
Biotechnology and Bioengineering
|January 26, 2016
Summary
We developed a new bead display method to find ligation enzymes, enabling screening of 10(12) variants. This identified a novel sortase A enzyme with enhanced activity for potential intracellular labeling and synthetic biology applications.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Synthetic Biology
Background:
- Existing enzyme evolution methods have limitations in library size, turnover efficiency, and substrate specificity.
- There is a need for robust selection strategies to identify novel enzymes with improved catalytic properties and cellular compatibility.
Purpose of the Study:
- To develop a generalized in vitro compartmentalization (IVC)-based bead display selection strategy for identifying enzymes capable of ligation reactions.
- To overcome limitations of existing methods by enabling larger library screening and multiple turnover selection.
- To isolate and characterize novel enzymes with enhanced catalytic activity and intracellular functionality.
Main Methods:
- Developed a generalized in vitro compartmentalization (IVC)-based bead display selection strategy.
- Screened libraries up to 10(12) protein variants by overloading beads with up to 10(4) unique mutants.
- Isolated and characterized a variant of sortase A from Staphylococcus aureus.
Main Results:
- The developed IVC-based bead display strategy allows for screening of significantly larger libraries compared to previous methods.
- Isolated a variant of Staphylococcus aureus sortase A with a 114-fold enhancement in kcat/KM compared to wild-type, in the absence of calcium.
- The selected variant demonstrated improved resistance to cell lysate inhibition and exhibited intracellular activity in eukaryotic cells.
Conclusions:
- The novel IVC-based bead display strategy is effective for identifying enzymes with desired catalytic functions, overcoming limitations of prior techniques.
- The engineered sortase A variant possesses superior catalytic efficiency and stability, making it suitable for advanced applications.
- The intracellular activity of the variant opens new possibilities for in vivo labeling and synthetic biology applications within eukaryotic cells.
