Solubilization and humanization of paraoxonase-1

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.

Figure 1

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.

Figure 2

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.

Figure 3

PON1 solubilizing mutations. (a) The surface of G2E6 is shown, with hydrophobic amino acids (VGMCILYFW) shown in red. Residues 1–15 are not resolved in the X-ray crystal structure, but the ΔN-huPON1 variant removed residues 4–17. (b) The positions modified in the ΔHDL-huPON1 variant are shown in spheres. These residues compose much of the hydrophobic surface patch near the N-terminus evident in (a). The Ca²⁺ ions are shown as pink spheres, and a phosphate bound in the presumed active site is shown in orange sticks. (c) The 59 positions that differ between huPON1 and G2E6 are shown; the positions that were modified in g2e6p-huPON1 are spheres, and the other 43 positions are sticks. Position 166, which was modified because of its proximity of 192, is noted. Rendered from PDB ID: 1V04 with PyMOL.

Figure 4

GFP-fusion screening for solubility. The fluorescence level is shown for normalized numbers of cells in PBS with frGFP fusions of the PON1 variants. Results from multiple trials typically varied by less than 10%. Negative control cells contained no frGFP fusion. For clarity, the constructs are listed in the legend in the same order from top to bottom as the maxima of the emission spectra.

Figure 5

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.

Figure 6

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

Figure 7

Stability of huPON1 variants. The residual activities against phenyl acetate after 10 min of incubation at the indicated temperatures are shown. The residual activity after incubation at 20°C was taken as 100%. (a) huPON1 and ΔN-huPON1; (b) ΔHDL-huPON1 and ΔN-ΔHDL-huPON1; (c) g2e6p-huPON1 and ΔN-g2e6p-huPON1.

Figure 8

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.