Limits...
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

Humanization of 4E9. (a) The g2e6p mutations are shown in green spheres; the three additional mutations M55L, A126T, and V206T, are shown in blue spheres; the 4E9 mutations (with respect to G3C9) are shown in red spheres. (b) Thermal inactivation of hum-4E9 produced with and without MBP fusion is compared to huPON1 and 4E9. Residual activity here was from EMP hydrolysis rather than phenyl acetate because the H115W mutation renders PON1 inactive against phenyl acetate.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3376767&req=5

fig8: Humanization of 4E9. (a) The g2e6p mutations are shown in green spheres; the three additional mutations M55L, A126T, and V206T, are shown in blue spheres; the 4E9 mutations (with respect to G3C9) are shown in red spheres. (b) Thermal inactivation of hum-4E9 produced with and without MBP fusion is compared to huPON1 and 4E9. Residual activity here was from EMP hydrolysis rather than phenyl acetate because the H115W mutation renders PON1 inactive against phenyl acetate.

Mentions: We wished to test whether we could produce a solubilized huPON1 with significant activity toward an OP compound, effectively “humanizing” an engineered PON1. The g2e6p-huPON1 variant appeared to afford the best combination of solubility, activity, and stability from among our original variants, so we elected to use those mutations to humanize an engineered PON1. Gupta and colleagues recently reported the engineering of a PON1 variant called 4E9 that has significant activity toward the cyclosarin (GF) analog CMP and notably increased activity against the more toxic SP enantiomorph of CMP [15]. It also has significant activity against authentic GF, as determined from an AChE inactivation assay in which GF is generated in situ at low concentrations. 4E9 is derived from G3C9, with the mutations L69G S111T H115W H134R F222S T332S, all of which are in the presumed active site except for S111T. Therefore, we generated a variant of huPON1 with surface solubilizing mutations derived from G2E6 (akin to the g2e6p-huPON1 described above and in Figure 1) and the 4E9 mutations obtained during directed evolution. The resulting variant, hum-4E9 (Figure 8), has two additional nonpolar-to-polar mutations that we did not elect to make in the g2e6p-huPON1: A126T and V206T. As an additional test of the solubilization afforded by the surface polar mutations, we not only expressed hum-4E9 as an MBP fusion with C-terminal 6 × his tag, but we also produced it with no fusion partner (with a C-terminal 6 × His tag only, which is also present in G3C9 and 4E9). The MBP fusion of hum-4E9 was purified in similar yield to the other MBP fusions of the huPON1 variants and G2E6. The unfused hum-4E9 was produced in about 5-fold lower yield than this and approximately 25-fold lower yield than 4E9 itself.


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)

Humanization of 4E9. (a) The g2e6p mutations are shown in green spheres; the three additional mutations M55L, A126T, and V206T, are shown in blue spheres; the 4E9 mutations (with respect to G3C9) are shown in red spheres. (b) Thermal inactivation of hum-4E9 produced with and without MBP fusion is compared to huPON1 and 4E9. Residual activity here was from EMP hydrolysis rather than phenyl acetate because the H115W mutation renders PON1 inactive against phenyl acetate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: Humanization of 4E9. (a) The g2e6p mutations are shown in green spheres; the three additional mutations M55L, A126T, and V206T, are shown in blue spheres; the 4E9 mutations (with respect to G3C9) are shown in red spheres. (b) Thermal inactivation of hum-4E9 produced with and without MBP fusion is compared to huPON1 and 4E9. Residual activity here was from EMP hydrolysis rather than phenyl acetate because the H115W mutation renders PON1 inactive against phenyl acetate.
Mentions: We wished to test whether we could produce a solubilized huPON1 with significant activity toward an OP compound, effectively “humanizing” an engineered PON1. The g2e6p-huPON1 variant appeared to afford the best combination of solubility, activity, and stability from among our original variants, so we elected to use those mutations to humanize an engineered PON1. Gupta and colleagues recently reported the engineering of a PON1 variant called 4E9 that has significant activity toward the cyclosarin (GF) analog CMP and notably increased activity against the more toxic SP enantiomorph of CMP [15]. It also has significant activity against authentic GF, as determined from an AChE inactivation assay in which GF is generated in situ at low concentrations. 4E9 is derived from G3C9, with the mutations L69G S111T H115W H134R F222S T332S, all of which are in the presumed active site except for S111T. Therefore, we generated a variant of huPON1 with surface solubilizing mutations derived from G2E6 (akin to the g2e6p-huPON1 described above and in Figure 1) and the 4E9 mutations obtained during directed evolution. The resulting variant, hum-4E9 (Figure 8), has two additional nonpolar-to-polar mutations that we did not elect to make in the g2e6p-huPON1: A126T and V206T. As an additional test of the solubilization afforded by the surface polar mutations, we not only expressed hum-4E9 as an MBP fusion with C-terminal 6 × his tag, but we also produced it with no fusion partner (with a C-terminal 6 × His tag only, which is also present in G3C9 and 4E9). The MBP fusion of hum-4E9 was purified in similar yield to the other MBP fusions of the huPON1 variants and G2E6. The unfused hum-4E9 was produced in about 5-fold lower yield than this and approximately 25-fold lower yield than 4E9 itself.

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