Comparative study to develop a single method for retrieving wide class of recombinant proteins from classical...

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Expression

Background:

• Inclusion bodies (IBs) formation is a major challenge in heterologous protein expression.
• Existing methods for recovering proteins from IBs are often protein-specific and inefficient, especially for harsh IBs.

Purpose of the Study:

• To develop a novel, broadly applicable strategy for recovering active recombinant proteins from harsh inclusion bodies.
• To improve the efficiency and yield of active protein recovery compared to traditional methods.

Main Methods:

• Integration of mild solubilization techniques, including low sarkosyl concentration (0.05–0.1%).
• Application of a slow freeze (–1 °C/min) and fast thaw cycle.
• Utilizing a single buffer dilution method for protein refolding across different recombinant protein sub-classes.

Main Results:

• The proposed method significantly enhanced protein solubility and preserved the integrity of solubilized proteins.
• Active protein recovery was substantially higher compared to conventional solubilization and refolding approaches.
• The mild solubilization process effectively restored native-like protein conformation.

Conclusions:

• This integrated strategy offers a robust and milder approach for recovering active proteins from challenging inclusion bodies.
• The method demonstrates broad applicability across various recombinant protein sub-classes, overcoming limitations of existing techniques.