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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

Alignment of PON1 variants. All differences are noted with respect to the human PON1 sequences (Q192/M55 polymorph); the 4E9 sequence and the similar rabbit PON1 sequence are shown in full for reference. Differences between G3C9 and 4E9 are noted in red in the 4E9 sequence. -: deletion.
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Related In: Results  -  Collection


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fig1: Alignment of PON1 variants. All differences are noted with respect to the human PON1 sequences (Q192/M55 polymorph); the 4E9 sequence and the similar rabbit PON1 sequence are shown in full for reference. Differences between G3C9 and 4E9 are noted in red in the 4E9 sequence. -: deletion.

Mentions: As a result of the physiological and toxicological correlations with increased PON1 activity, there is great motivation to develop PON1 as a therapeutic agent. There are significant difficulties with this: PON1 has only moderate solubility; it has three Cys residues including two forming a disulfide bond, and it is glycosylated [16]. Human PON1 (huPON1) is very difficult to produce in soluble, folded form in E. coli. Large-scale fermentation has been used to produce soluble huPON1 successfully in E. coli, but in poor yields for pharmaceutical production [14]. This motivated Aharoni and colleagues to generate a chimeric mammalian PON1 by DNA shuffling of mouse, rat, rabbit, and human PON1 isoforms, resulting in a variant called G2E6 that could be expressed when fused to the C-terminus of thioredoxin in good yields in the soluble fraction of E. coli (Figure 1) [17]. The crystal structure of G2E6 was solved, revealing it to be a six-bladed β-propeller protein bound to two Ca2+ ions, one of which appears to play a more structural role and one of which is located in what is presumed to be the active-site pocket [16]. A further generation of DNA shuffling and selection yielded G3C9 PON1, which can be expressed in significant amounts in the soluble fraction of E. coli without a fusion partner (although it bears a C-terminal hexahistidine tag). Both of the proteins have greatest sequence similarity to the rabbit isoform of PON1, and they differ by 58-59 (G2E6) and 50-51 (G3C9) amino acids from huPON1, depending on the polymorph, mostly on the surface and essentially not at all in the putative active site. (Human PON1 is either Leu or Met at 55, while rabbit is only known to be Leu at this position).


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)

Alignment of PON1 variants. All differences are noted with respect to the human PON1 sequences (Q192/M55 polymorph); the 4E9 sequence and the similar rabbit PON1 sequence are shown in full for reference. Differences between G3C9 and 4E9 are noted in red in the 4E9 sequence. -: deletion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Alignment of PON1 variants. All differences are noted with respect to the human PON1 sequences (Q192/M55 polymorph); the 4E9 sequence and the similar rabbit PON1 sequence are shown in full for reference. Differences between G3C9 and 4E9 are noted in red in the 4E9 sequence. -: deletion.
Mentions: As a result of the physiological and toxicological correlations with increased PON1 activity, there is great motivation to develop PON1 as a therapeutic agent. There are significant difficulties with this: PON1 has only moderate solubility; it has three Cys residues including two forming a disulfide bond, and it is glycosylated [16]. Human PON1 (huPON1) is very difficult to produce in soluble, folded form in E. coli. Large-scale fermentation has been used to produce soluble huPON1 successfully in E. coli, but in poor yields for pharmaceutical production [14]. This motivated Aharoni and colleagues to generate a chimeric mammalian PON1 by DNA shuffling of mouse, rat, rabbit, and human PON1 isoforms, resulting in a variant called G2E6 that could be expressed when fused to the C-terminus of thioredoxin in good yields in the soluble fraction of E. coli (Figure 1) [17]. The crystal structure of G2E6 was solved, revealing it to be a six-bladed β-propeller protein bound to two Ca2+ ions, one of which appears to play a more structural role and one of which is located in what is presumed to be the active-site pocket [16]. A further generation of DNA shuffling and selection yielded G3C9 PON1, which can be expressed in significant amounts in the soluble fraction of E. coli without a fusion partner (although it bears a C-terminal hexahistidine tag). Both of the proteins have greatest sequence similarity to the rabbit isoform of PON1, and they differ by 58-59 (G2E6) and 50-51 (G3C9) amino acids from huPON1, depending on the polymorph, mostly on the surface and essentially not at all in the putative active site. (Human PON1 is either Leu or Met at 55, while rabbit is only known to be Leu at this position).

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