Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins.

Area of Science:

• Biochemistry
• Biotechnology
• Protein Science

Background:

• Amyloid formation is implicated in various diseases.
• Surface activity can influence protein self-assembly.
• Efficient isotope labeling is crucial for structural and functional studies of proteins.

Purpose of the Study:

• To develop a cost-effective isotope labeling procedure for amyloidogenic proteins.
• To optimize fed-batch fermentation for enhanced isotope incorporation.
• To assess the efficiency of the developed method using a miniprotein model.

Main Methods:

• Fed-batch fermentation in E. coli using minimal media.
• Controlled feeding of 15N-NH4Cl and 13C-D-Glc.
• Optimization of nutrient feeding ratios (1:5 NH4Cl:D-Glc) and feeding schemes.
• Characterization of protein structure and amyloid formation using PM-IRRAS and AFM.

Main Results:

• Achieved 1.5- to 5-fold increase in miniprotein yields.
• Reduced labeling costs significantly compared to flask methods.
• Demonstrated efficient incorporation of 15N, 13C, and 15N/13C isotopes.
• Confirmed E5 miniprotein's amyloidogenic potential at the air-water interface.

Conclusions:

• The optimized fed-batch fermentation method is highly efficient for cost-effective isotope labeling.
• This approach is suitable for producing labeled amyloidogenic proteins, even complex sequences.
• The developed protocols offer a scalable and economical solution for protein labeling in research.