Novel Self-Cleaving Affinity Purification Method for Cellular Membrane-Associated Recombinant Paraoxonase-1 (rePON1)...

Area of Science:

• Biochemistry
• Protein Chemistry
• Enzyme Engineering

Background:

• Mammalian paraoxonase-1 (PON1) is a calcium-dependent hydrolytic enzyme with therapeutic potential.
• PON1's membrane association causes aggregation and instability issues during purification.
• Existing methods for bacterial expression of PON1 face challenges with proteolytic tag removal and detergent dependency.

Purpose of the Study:

• To develop a novel affinity purification strategy for recombinant PON1 (rePON1).
• To overcome limitations in solubility, stability, and tag removal associated with PON1 purification.
• To enable efficient bioanalytical characterization of tagless, native PON1.

Main Methods:

• Utilized a novel strategy combining two solubility-enhancing fusion partners with the iCapTag™ self-removing affinity tag.
• Conducted the entire purification process in the presence of detergent.
• Optimized purification conditions, including detergent type and pH, for rePON1 solubilization and stabilization.

Main Results:

• Successfully achieved solubilization and stabilization of rePON1 at room temperature.
• Obtained tagless, native PON1, confirming expected catalytic efficiency and molecular weight.
• Achieved >95% host-cell protein impurity removal and >99.9% host cell DNA clearance in a single affinity column step.

Conclusions:

• The developed method offers a versatile and efficient alternative for purifying recombinant membrane-associated proteins.
• This approach facilitates facile tag removal and characterization of challenging proteins.
• The strategy is applicable to producing other recombinant proteins requiring specific buffer conditions.