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Solubilization and humanization of paraoxonase-1.

Sarkar M, Harsch CK, Matic GT, Hoffman K, Norris JR, Otto TC, Lenz DE, Cerasoli DM, Magliery TJ - J Lipids (2012)

Bottom Line: Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1.All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most.Based on the G2E6 polar mutations, we "humanized" an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Paraoxonase-1 (PON1) is a serum protein, the activity of which is related to susceptibility to cardiovascular disease and intoxication by organophosphorus (OP) compounds. It may also be involved in innate immunity, and it is a possible lead molecule in the development of a catalytic bioscavenger of OP pesticides and nerve agents. Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1. We examined the effect on the solubility, activity, and stability of three sets of mutations designed to solubilize huPON1 with fewer overall changes: deletion of the N-terminal leader, polar mutations in the putative HDL binding site, and selection of the subset of residues that became more polar in going from huPON1 to G2E6. All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most. Based on the G2E6 polar mutations, we "humanized" an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence.

No MeSH data available.


Related in: MedlinePlus

Stability of huPON1 variants. The residual activities against phenyl acetate after 10 min of incubation at the indicated temperatures are shown. The residual activity after incubation at 20°C was taken as 100%. (a) huPON1 and ΔN-huPON1; (b) ΔHDL-huPON1 and ΔN-ΔHDL-huPON1; (c) g2e6p-huPON1 and ΔN-g2e6p-huPON1.
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fig7: Stability of huPON1 variants. The residual activities against phenyl acetate after 10 min of incubation at the indicated temperatures are shown. The residual activity after incubation at 20°C was taken as 100%. (a) huPON1 and ΔN-huPON1; (b) ΔHDL-huPON1 and ΔN-ΔHDL-huPON1; (c) g2e6p-huPON1 and ΔN-g2e6p-huPON1.

Mentions: We were also interested in the effects of each of these solubilizing sets of mutations on the stabilities of the resulting proteins. Because PON1 denatures irreversibly upon heating, thermal inactivation is a good measure of the relative stability of the variants [33]. With phenyl acetate activity as the read-out, the T1/2 for huPON1 was roughly 55–60°C (Figure 7). The ΔN-huPON1 was increased slightly in stability, to about 60°C. Both g2e6p-huPON1 and ΔN-g2e6p-huPON1 have T1/2 values close to 55°C, and both ΔHDL-huPON1 and ΔN-ΔHDL-huPON1 have T1/2 values close to 40°C. A similar experiment using EMP as the substrate produced similar results, with huPON1 showing a midpoint in the inactivation curve around 55°C and ΔN-huPON1 and g2e6p-huPON around 50°C. Overall we conclude that the N-terminal deletion had little effect on the stability, the g2e6p mutations reduced the stability of huPON1 slightly, and the ΔHDL mutations reduced it significantly.


Solubilization and humanization of paraoxonase-1.

Sarkar M, Harsch CK, Matic GT, Hoffman K, Norris JR, Otto TC, Lenz DE, Cerasoli DM, Magliery TJ - J Lipids (2012)

Stability of huPON1 variants. The residual activities against phenyl acetate after 10 min of incubation at the indicated temperatures are shown. The residual activity after incubation at 20°C was taken as 100%. (a) huPON1 and ΔN-huPON1; (b) ΔHDL-huPON1 and ΔN-ΔHDL-huPON1; (c) g2e6p-huPON1 and ΔN-g2e6p-huPON1.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376767&req=5

fig7: Stability of huPON1 variants. The residual activities against phenyl acetate after 10 min of incubation at the indicated temperatures are shown. The residual activity after incubation at 20°C was taken as 100%. (a) huPON1 and ΔN-huPON1; (b) ΔHDL-huPON1 and ΔN-ΔHDL-huPON1; (c) g2e6p-huPON1 and ΔN-g2e6p-huPON1.
Mentions: We were also interested in the effects of each of these solubilizing sets of mutations on the stabilities of the resulting proteins. Because PON1 denatures irreversibly upon heating, thermal inactivation is a good measure of the relative stability of the variants [33]. With phenyl acetate activity as the read-out, the T1/2 for huPON1 was roughly 55–60°C (Figure 7). The ΔN-huPON1 was increased slightly in stability, to about 60°C. Both g2e6p-huPON1 and ΔN-g2e6p-huPON1 have T1/2 values close to 55°C, and both ΔHDL-huPON1 and ΔN-ΔHDL-huPON1 have T1/2 values close to 40°C. A similar experiment using EMP as the substrate produced similar results, with huPON1 showing a midpoint in the inactivation curve around 55°C and ΔN-huPON1 and g2e6p-huPON around 50°C. Overall we conclude that the N-terminal deletion had little effect on the stability, the g2e6p mutations reduced the stability of huPON1 slightly, and the ΔHDL mutations reduced it significantly.

Bottom Line: Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1.All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most.Based on the G2E6 polar mutations, we "humanized" an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Paraoxonase-1 (PON1) is a serum protein, the activity of which is related to susceptibility to cardiovascular disease and intoxication by organophosphorus (OP) compounds. It may also be involved in innate immunity, and it is a possible lead molecule in the development of a catalytic bioscavenger of OP pesticides and nerve agents. Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1. We examined the effect on the solubility, activity, and stability of three sets of mutations designed to solubilize huPON1 with fewer overall changes: deletion of the N-terminal leader, polar mutations in the putative HDL binding site, and selection of the subset of residues that became more polar in going from huPON1 to G2E6. All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most. Based on the G2E6 polar mutations, we "humanized" an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence.

No MeSH data available.


Related in: MedlinePlus