Construction and flow cytometric screening of targeted enzyme libraries

Area of Science:

• Enzymology
• Molecular Biology
• Biotechnology

Background:

• Cell-surface proteases play crucial roles in biological processes.
• Modifying protease substrate specificity is essential for various biotechnological applications.
• Escherichia coli outer membrane protease OmpT serves as a model for studying protease engineering.

Purpose of the Study:

• To develop a robust methodology for constructing targeted gene libraries to alter protease substrate specificity.
• To enable systematic screening of enzyme variants with modified functions using flow cytometry.
• To provide a protocol for partial multi-site saturation library construction for enzymes with unelucidated substrate-binding pockets.

Main Methods:

• Gene assembly using oligonucleotides for targeted library construction.
• Systematic generation of increasingly complex enzyme libraries.
• Screening of engineered variants using fluorescence-activated cell sorting (FACS).

Main Results:

• A methodology for targeted library construction to modify protease substrate specificity was established.
• The protocol is adaptable to various enzymes, particularly those with known active site residues.
• Successful application demonstrated using the outer membrane protease OmpT in Escherichia coli.

Conclusions:

• The described gene assembly and FACS screening approach provides an efficient route to engineer cell-surface protease specificity.
• The methodology facilitates the creation of novel enzyme variants with tailored substrate specificities.
• This technique offers a valuable tool for advancing protease engineering in biotechnology and synthetic biology.