Screening for recombinant glutathione transferases active with monochlorobimane

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Glutathione transferases (GSTs) are crucial enzymes involved in detoxification and cellular defense.
• Screening large libraries of mutated GSTs for catalytic activity is challenging.
• Developing efficient methods to identify active enzyme variants is essential for protein engineering.

Purpose of the Study:

• To develop a rapid and facile colony assay for identifying catalytically active glutathione transferases (GSTs).
• To enable the screening of combinatorial cDNA libraries of mutated GSTs expressed in Escherichia coli.
• To facilitate the isolation and characterization of active recombinant GST clones.

Main Methods:

• Development of a colony assay based on the conjugation of glutathione (GSH) with the fluorogenic substrate monochlorobimane (MCB).
• Expression of mutated glutathione transferases (GSTs) in Escherichia coli.
• Visual screening of bacterial colonies for fluorescence under long-wavelength UV light, indicating GST activity.

Main Results:

• The assay successfully identified active glutathione transferases (GSTs) in bacterial colonies.
• Fluorescence was observed instantly in colonies containing active GSTs catalyzing the GSH-MCB conjugation.
• Eleven human and one rat GSTs were tested, with all except human GST T1-1 yielding fluorescent colonies.

Conclusions:

• The developed colony assay is broadly applicable for identifying active glutathione transferases (GSTs).
• This method is effective for screening both wild-type enzyme subclones and mutant libraries.
• The assay provides a rapid and efficient tool for enzyme discovery and engineering.