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

Purification of huPON1 variants from E. coli. (a) SDS-PAGE of purification from His6-MBP-PON1 fusions from the pHMT plasmid using NiNTA chromatography. (1) ΔN-huPON1; (2) ΔHDL-huPON1; (3) ΔN-ΔHDL-huPON1; (4) g2e6p-huPON1. (b) Blot of SDS-PAGE of PON1 variants as His6-MBP-PON1 fusions with HisProbe-HRP (Pierce), demonstrating that many of the smaller proteins bear the 6 × His tag and are likely proteolytic fragments. (1) ΔHDL-huPON1 lysate; (2) and (3), purified ΔHDL-huPON1; (4) cleaved MBP; (5) purified g2e6p-huPON1; (6) purified ΔN-huPON1. (c) SDS-PAGE of purified proteins from the MBP-PON1-His6 constructs in pET11a with coexpression of DnaK/DnaJ/GrpE from pKJE7 (Takara Bioscience). (1) huPON1; (2) ΔN-huPON1; (3) ΔHDL-huPON1; (4) ΔN-ΔHDL-huPON1; (5) g2e6p-huPON1; (6) ΔN-g2e6p-huPON1.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3376767&req=5

fig5: Purification of huPON1 variants from E. coli. (a) SDS-PAGE of purification from His6-MBP-PON1 fusions from the pHMT plasmid using NiNTA chromatography. (1) ΔN-huPON1; (2) ΔHDL-huPON1; (3) ΔN-ΔHDL-huPON1; (4) g2e6p-huPON1. (b) Blot of SDS-PAGE of PON1 variants as His6-MBP-PON1 fusions with HisProbe-HRP (Pierce), demonstrating that many of the smaller proteins bear the 6 × His tag and are likely proteolytic fragments. (1) ΔHDL-huPON1 lysate; (2) and (3), purified ΔHDL-huPON1; (4) cleaved MBP; (5) purified g2e6p-huPON1; (6) purified ΔN-huPON1. (c) SDS-PAGE of purified proteins from the MBP-PON1-His6 constructs in pET11a with coexpression of DnaK/DnaJ/GrpE from pKJE7 (Takara Bioscience). (1) huPON1; (2) ΔN-huPON1; (3) ΔHDL-huPON1; (4) ΔN-ΔHDL-huPON1; (5) g2e6p-huPON1; (6) ΔN-g2e6p-huPON1.

Mentions: We wished to purify the huPON1 solubilized variants to measure their activity and stability, as well as to verify their soluble expression. However, we found that the frGFP fusions were expressed at such low levels that it was inconvenient to work with them. As a result, we recloned the constructs, fusing a hexahistidine tag and maltose-binding protein to the N-terminus of the huPON1 variants. When these variants were purified by NiNTA affinity chromatography, they appeared to copurify with a significant number of smaller proteins at reduced but significant levels (Figure 5). Note that, for the MBP fusions, the amount of soluble protein captured in the purification was greatest for ΔN-ΔHDL-huPON1 and then ΔHDL-huPON1; g2e6p-huPON1 and ΔN-huPON1 were purified in comparable lower amounts, consistent with the screening data. It proved difficult to purify the proteins with increasingly stringent washes, which made us suspect that the other bands on the SDS-PAGE gel were truncation products of the full-length constructs. A blot with the anti-His6 reagent HisProbe-HRP confirmed that the smaller proteins contained a hexahistidine tag.


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)

Purification of huPON1 variants from E. coli. (a) SDS-PAGE of purification from His6-MBP-PON1 fusions from the pHMT plasmid using NiNTA chromatography. (1) ΔN-huPON1; (2) ΔHDL-huPON1; (3) ΔN-ΔHDL-huPON1; (4) g2e6p-huPON1. (b) Blot of SDS-PAGE of PON1 variants as His6-MBP-PON1 fusions with HisProbe-HRP (Pierce), demonstrating that many of the smaller proteins bear the 6 × His tag and are likely proteolytic fragments. (1) ΔHDL-huPON1 lysate; (2) and (3), purified ΔHDL-huPON1; (4) cleaved MBP; (5) purified g2e6p-huPON1; (6) purified ΔN-huPON1. (c) SDS-PAGE of purified proteins from the MBP-PON1-His6 constructs in pET11a with coexpression of DnaK/DnaJ/GrpE from pKJE7 (Takara Bioscience). (1) huPON1; (2) ΔN-huPON1; (3) ΔHDL-huPON1; (4) ΔN-ΔHDL-huPON1; (5) g2e6p-huPON1; (6) ΔN-g2e6p-huPON1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Purification of huPON1 variants from E. coli. (a) SDS-PAGE of purification from His6-MBP-PON1 fusions from the pHMT plasmid using NiNTA chromatography. (1) ΔN-huPON1; (2) ΔHDL-huPON1; (3) ΔN-ΔHDL-huPON1; (4) g2e6p-huPON1. (b) Blot of SDS-PAGE of PON1 variants as His6-MBP-PON1 fusions with HisProbe-HRP (Pierce), demonstrating that many of the smaller proteins bear the 6 × His tag and are likely proteolytic fragments. (1) ΔHDL-huPON1 lysate; (2) and (3), purified ΔHDL-huPON1; (4) cleaved MBP; (5) purified g2e6p-huPON1; (6) purified ΔN-huPON1. (c) SDS-PAGE of purified proteins from the MBP-PON1-His6 constructs in pET11a with coexpression of DnaK/DnaJ/GrpE from pKJE7 (Takara Bioscience). (1) huPON1; (2) ΔN-huPON1; (3) ΔHDL-huPON1; (4) ΔN-ΔHDL-huPON1; (5) g2e6p-huPON1; (6) ΔN-g2e6p-huPON1.
Mentions: We wished to purify the huPON1 solubilized variants to measure their activity and stability, as well as to verify their soluble expression. However, we found that the frGFP fusions were expressed at such low levels that it was inconvenient to work with them. As a result, we recloned the constructs, fusing a hexahistidine tag and maltose-binding protein to the N-terminus of the huPON1 variants. When these variants were purified by NiNTA affinity chromatography, they appeared to copurify with a significant number of smaller proteins at reduced but significant levels (Figure 5). Note that, for the MBP fusions, the amount of soluble protein captured in the purification was greatest for ΔN-ΔHDL-huPON1 and then ΔHDL-huPON1; g2e6p-huPON1 and ΔN-huPON1 were purified in comparable lower amounts, consistent with the screening data. It proved difficult to purify the proteins with increasingly stringent washes, which made us suspect that the other bands on the SDS-PAGE gel were truncation products of the full-length constructs. A blot with the anti-His6 reagent HisProbe-HRP confirmed that the smaller proteins contained a hexahistidine tag.

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