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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

Structures of the substrates used in this study. CMP, EMP, and GF are shown as the SP enantiomorphs.
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fig6: Structures of the substrates used in this study. CMP, EMP, and GF are shown as the SP enantiomorphs.

Mentions: The activity of our engineered PON1 variants against phenyl acetate and the OP compounds paraoxon and EMP was determined and compared with huPON1 and G2E6 (see Figure 6 for structures of the substrates). Because the activity levels were modest and initial trials suggested that the Km values were likely to be near the top of the concentration range that could be tested with each substrate, specific activities are reported for some substrates (Table 1).


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)

Structures of the substrates used in this study. CMP, EMP, and GF are shown as the SP enantiomorphs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Structures of the substrates used in this study. CMP, EMP, and GF are shown as the SP enantiomorphs.
Mentions: The activity of our engineered PON1 variants against phenyl acetate and the OP compounds paraoxon and EMP was determined and compared with huPON1 and G2E6 (see Figure 6 for structures of the substrates). Because the activity levels were modest and initial trials suggested that the Km values were likely to be near the top of the concentration range that could be tested with each substrate, specific activities are reported for some substrates (Table 1).

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