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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

Schematics of the fusions used in this study. PON1, paraoxonase-1 variant; TEV, TEV protease site; frGFP, folding reporter GFP; His6, hexahistidine tag; MBP, maltose-binding protein.
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Related In: Results  -  Collection


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fig2: Schematics of the fusions used in this study. PON1, paraoxonase-1 variant; TEV, TEV protease site; frGFP, folding reporter GFP; His6, hexahistidine tag; MBP, maltose-binding protein.

Mentions: The frGFP gene was generated in our lab from the genes for GFPuv [26] and EGFP [27] by overlap PCR, resulting in a GFP with mutations F64L S65T F99S M153T V163A. The frGFP gene was PCR amplified with primers coding for a 6 × His tag and an AatII site at the 3′ end and an EcoRI site at the 5′ end. Wild-type human PON1 (Q192/M55) was PCR amplified from a mammalian expression vector, pcDNA3. The oligonucleotide (Sigma Genosys, The Woodlands, TX) primers 5′-AATAATTATC ATATGGCTAA GCTGATTGCG CTCACCC-3′ with an NdeI site, and 5′-ATAATGAATT CGCCGCTGCT TCCGCTCTGA AAATACAGAT TCTCACCGCC GGTACCGAGT TCGCAGTAAA GAGCTTTGTG AAACAC-3′ coding for a KpnI site, TEV protease (ENLYFQG) site, linker (GSSG) and EcoRI site, were used for amplification. A fusion of huPON1-(KpnI-)TEV-Linker-(EcoRI-)frGFP (Figure 2) was cloned into a pET11a vector between the NdeI and AatII sites using a three-piece ligation. For reference, the G2E6 gene was analogously cloned into this construct (the gene was kindly provided by Dan Tawfik, Weizmann Institute of Science). The sequence of the fusion construct was confirmed by DNA sequencing (Genewiz, South Plainfield, NJ).


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)

Schematics of the fusions used in this study. PON1, paraoxonase-1 variant; TEV, TEV protease site; frGFP, folding reporter GFP; His6, hexahistidine tag; MBP, maltose-binding protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Schematics of the fusions used in this study. PON1, paraoxonase-1 variant; TEV, TEV protease site; frGFP, folding reporter GFP; His6, hexahistidine tag; MBP, maltose-binding protein.
Mentions: The frGFP gene was generated in our lab from the genes for GFPuv [26] and EGFP [27] by overlap PCR, resulting in a GFP with mutations F64L S65T F99S M153T V163A. The frGFP gene was PCR amplified with primers coding for a 6 × His tag and an AatII site at the 3′ end and an EcoRI site at the 5′ end. Wild-type human PON1 (Q192/M55) was PCR amplified from a mammalian expression vector, pcDNA3. The oligonucleotide (Sigma Genosys, The Woodlands, TX) primers 5′-AATAATTATC ATATGGCTAA GCTGATTGCG CTCACCC-3′ with an NdeI site, and 5′-ATAATGAATT CGCCGCTGCT TCCGCTCTGA AAATACAGAT TCTCACCGCC GGTACCGAGT TCGCAGTAAA GAGCTTTGTG AAACAC-3′ coding for a KpnI site, TEV protease (ENLYFQG) site, linker (GSSG) and EcoRI site, were used for amplification. A fusion of huPON1-(KpnI-)TEV-Linker-(EcoRI-)frGFP (Figure 2) was cloned into a pET11a vector between the NdeI and AatII sites using a three-piece ligation. For reference, the G2E6 gene was analogously cloned into this construct (the gene was kindly provided by Dan Tawfik, Weizmann Institute of Science). The sequence of the fusion construct was confirmed by DNA sequencing (Genewiz, South Plainfield, NJ).

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