Solubilization and humanization of paraoxonase-1

Area of Science:

• Biochemistry
• Enzyme Engineering
• Toxicology

Background:

• Paraoxonase-1 (PON1) is a crucial serum protein linked to cardiovascular disease and organophosphorus (OP) compound toxicity.
• Its potential role in innate immunity and as a bioscavenger for OP pesticides and nerve agents is significant.
• Recombinant human PON1 (huPON1) expression in E. coli yields predominantly insoluble protein, necessitating strategies for soluble variant development.

Purpose of the Study:

• To engineer more soluble variants of huPON1 with fewer mutations than existing G2E6 variants.
• To assess the impact of specific mutation strategies on solubility, activity, and stability.
• To develop a humanized PON1 variant with retained high catalytic activity against cyclosarin (GF).

Main Methods:

• Investigated three sets of mutations: N-terminal leader deletion, polar mutations in the HDL binding site, and selection of polar residues from the G2E6 variant.
• Evaluated solubility, enzymatic activity, and stability of the engineered huPON1 variants.
• Humanized a cyclosarin-active engineered PON1 variant using insights from G2E6 polar mutations.

Main Results:

• All three mutation strategies successfully increased huPON1 solubility.
• The HDL-binding site mutations yielded the greatest solubility increase but also significantly reduced activity and stability.
• The humanized variant retained high cyclosarin-degrading activity while exhibiting greater sequence similarity to native huPON1.

Conclusions:

• Targeted mutations can enhance the solubility of recombinant huPON1.
• Balancing solubility, activity, and stability is critical in enzyme engineering for bioscavenger applications.
• A humanized PON1 variant with high cyclosarinase activity and improved human sequence homology was successfully developed.