Folding Optimization In Vivo Uncovers New Chaperones

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Protein misfolding and aggregation are significant challenges in biotechnology and disease.
• The periplasm is a critical cellular compartment for protein folding and modification in bacteria.
• Existing methods for enhancing protein folding capacity in vivo are limited.

Purpose of the Study:

• To develop bacterial strains with improved periplasmic protein folding capacity.
• To identify novel proteins involved in enhancing protein folding.
• To create a selection system for engineering protein stabilization in bacteria.

Main Methods:

• A genetic selection strategy was employed, forcing bacterial survival through the folding of an unstable, aggregation-prone test protein.
• Bacterial variants with enhanced folding capacity were isolated and analyzed.
• Gene expression analysis was performed to identify overexpressed periplasmic proteins.

Main Results:

• Generated bacterial strains exhibiting enhanced periplasmic folding capacity.
• Observed increased soluble steady-state levels of the test protein in engineered strains.
• Identified overexpression of several periplasmic proteins, including OsmY, Ivy, DppA, OppA, and HdeB, with HdeB previously known as a chaperone.

Conclusions:

• A novel genetic selection method can effectively generate bacterial strains with enhanced protein folding capabilities.
• The engineered strains demonstrate improved capacity for stabilizing aggregation-prone proteins.
• This approach offers a powerful tool for creating "designer bacteria" selected for specific protein stabilization functions.