Aggregation of recombinant human interferon gamma: kinetics and structural transitions

Area of Science:

• Biochemistry
• Protein Science
• Biophysics

Background:

• Protein aggregation is a significant challenge in biotechnology, often leading to loss of protein function.
• Understanding general aggregation mechanisms, beyond specific stress factors, is crucial for protein stability.
• Recombinant human interferon-gamma (rhIFN-gamma) is a suitable model protein due to its aggregation propensity under mild conditions.

Purpose of the Study:

• To investigate the general aggregation mechanism of rhIFN-gamma.
• To characterize the structural and kinetic properties of protein aggregates formed under various mild denaturing stresses.
• To compare aggregates formed under denaturing stresses with those formed via salting out.

Main Methods:

• Spectroscopic techniques including infrared (IR) and circular dichroism (CD) spectroscopy were used to analyze protein structure.
• Kinetic studies were performed to determine the order of aggregation processes.
• Mild denaturing stresses (low denaturant, sub-Tm temperature, low pH) and salting out were employed to induce aggregation.

Main Results:

• Aggregates formed under mild denaturing stresses exhibited similar structures characterized by high intermolecular beta-sheet content and loss of alpha-helix.
• Both thermally and denaturant-induced aggregation followed first-order kinetics.
• Spectroscopic and kinetic data indicated aggregation proceeds through an intermediate with significant residual secondary structure.
• Protein aggregates formed by salting out retained a native-like secondary structure.

Conclusions:

• rhIFN-gamma aggregation under mild denaturing conditions involves an intermediate state with retained secondary structure.
• The aggregation pathway is consistent across different mild denaturing stresses.
• Protein aggregation significantly alters secondary structure, while salting out preserves it, offering insights into protein stabilization.