Activity-independent screening of secreted proteins using split GFP

Area of Science:

• Biotechnology
• Molecular Biology
• Microbial Engineering

Background:

• Efficient protein secretion is crucial for industrial applications, particularly in Gram-positive bacteria utilizing the Sec system.
• Current high-throughput screening methods for protein secretion often rely on photometric or fluorimetric assays measuring enzymatic activity.

Purpose of the Study:

• To develop and validate an alternative high-throughput screening method for quantifying secreted proteins using a split green fluorescent protein (GFP) assay.
• To assess the utility of the split GFP assay for monitoring both homologous and heterologous protein secretion in Bacillus subtilis.

Main Methods:

• Developed a split GFP assay for monitoring protein secretion in Bacillus subtilis.
• Analyzed the secretion of a homologous lipase and a heterologous cutinase using both GFP fluorescence and traditional enzyme activity assays.
• Screened a signal peptide library to identify variants that enhance the secretion of Bacillus subtilis swollenin EXLX1.

Main Results:

• The split GFP assay effectively quantified the secretion of both enzymatic and non-enzymatic proteins.
• Demonstrated successful application in analyzing secretion of homologous lipase and heterologous cutinase.
• Identified a signal peptide variant that increased secretion of swollenin EXLX1 by up to 5-fold.

Conclusions:

• The split GFP assay provides a robust and versatile alternative for high-throughput monitoring of protein secretion in Bacillus subtilis.
• This method facilitates protein engineering for optimizing industrial protein production.
• The developed assay is applicable to a wide range of secreted proteins, including enzymes and non-enzymatic proteins.