Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Overview

This article describes a method to monitor protein re-solubilization and refolding in vivo using a firefly luciferase-GFP fusion protein in Saccharomyces cerevisiae. The approach combines fluorescence microscopy and enzymatic assays to investigate the roles of proteostasis network components in protein quality control.

Key Study Components

Area of Science

• Cell Biology
• Protein Folding
• Biochemistry

Background

• Protein folding is critical for cellular function.
• Proteostasis networks maintain protein homeostasis.
• Fluorescence microscopy allows real-time observation of proteins.
• Enzymatic assays provide quantitative measures of protein activity.

Purpose of Study

• To investigate in vivo protein folding dynamics.
• To assess the efficiency of protein reactivation.
• To explore the roles of different proteostasis components.

Methods Used

• Transformation of yeast cells with a plasmid encoding F-F-L-G-F-P.
• Induction of F-F-L-G-F-P expression.
• Cycloheximide treatment to halt protein production.
• Measurement of luciferase activity before and after thermal denaturation.

Main Results

• Real-time monitoring of protein refolding was achieved.
• Fluorescence microscopy confirmed solubility of the fusion protein.
• Enzymatic assays quantified the reactivation efficiency.
• Multiple strains were analyzed for comparative results.

Conclusions

• The method effectively monitors protein folding in vivo.
• Fluorescence and enzymatic assays are complementary techniques.
• This approach enhances understanding of proteostasis mechanisms.

Frequently Asked Questions