Effects of solutes on solubilization and refolding of proteins from inclusion bodies with high hydrostatic pressure

Abstract

High hydrostatic pressure (HHP)-mediated solubilization and refolding of five inclusion bodies (IBs) produced from bacteria, three gram-negative binding proteins (GNBP1, GNBP2, and GNBP3) from Drosophila, and two phosphatases from human were investigated in combination of a redox-shuffling agent (2 mM DTT and 6 mM GSSG) and various additives. HHP (200 MPa) combined with the redox-shuffling agent resulted in solubilization yields of approximately 42%-58% from 1 mg/mL of IBs. Addition of urea (1 and 2 M), 2.5 M glycerol, L-arginine (0.5 M), Tween 20 (0.1 mM), or Triton X-100 (0.5 mM) significantly enhanced the solubilization yield for all proteins. However, urea, glycerol, and nonionic surfactants populated more soluble oligomeric species than monomeric species, whereas arginine dominantly induced functional monomeric species (approximately 70%-100%) to achieve refolding yields of approximately 55%-78% from IBs (1 mg/mL). Our results suggest that the combination of HHP with arginine is most effective in enhancing the refolding yield by preventing aggregation of partially folded intermediates populated during the refolding. Using the refolded proteins, the binding specificity of GNBP2 and GNBP3 was newly identified the same as with that of GNBP1, and the enzymatic activities of the two phosphatases facilitates their further characterization.

Figure 1.

Reducing (A) and nonreducing (B) SDS-PAGE analysis of the purified IBs: GNBP1 (lane 1), GNBP2 (lane 2), GNBP3 (lane 3), PTPRS (lane 4), and DUSP7 (lane 5). About 5 μg of each IB was analyzed on 12% SDS-PAGE under reducing and nonreducing conditions. The gels were stained with Coomassie blue. The molecular mass markers are indicated in kilodaltons.

Figure 2.

HHP mediated-solubilization yield of IBs (1 mg/mL) incubated under 200 MPa at 25°C for 24 h in the refolding buffer (50 mM Tris-Cl at pH 8.0, 150 mM NaCl, 1 mM EDTA, 2 mM DTT, 6 mM GSSG, 0.05% sodium azide) containing the various additives. The numbers below each column represent the buffer formulation as follows: (1) buffer alone; (2) 1 M urea; (3) 2 M urea; (4) 2.5 M glycerol; (5) 0.5 M arginine; (6) 0.1 mM Tween 20; (7) 0.5 mM Triton X-100. After decompression, samples were centrifuged (12,000g for 10 min) to remove insoluble aggregates and the supernatants were used for the total protein assay as described in the Materials and Methods in detail. The error bars represent the standard deviation for triplicate incubated samples.

Figure 3.

Representative size exclusion chromatogram for the recovered soluble GNBP1 (A) and PTPRS (B) from IBs (1 mg/mL) by HHP (200 MPa, 25°C, 24 h) in the refolding buffer containing the additives, as described in the legend for Figure 2 and also indicated in the legend box by different colors. The arrows indicate the elution position for monomeric species as M, for dimeric and/or trimeric species as O2, and for soluble aggregates (more than tetramer) as O1.

Figure 4.

Representative reducing (A) and nonreducing (B) SDS-PAGE analysis of the recovered soluble proteins from IBs (1 mg/mL) by HHP (200 MPa, 25°C, 24h) in the refolding buffer. GNBP1 (lane 1); GNBP2 (lane 2), GNBP3 (lane 3), PTPRS (lane 4), and DUSP7 (lane 5). The gels were stained with Coomassie blue.

Figure 5.

Functional binding assay of the recovered soluble GNBPs from their respective IBs (1 mg/mL) by HHP (200 MPa, 25°C, 24 h) in the refolding buffer containing 0.5 M arginine. The binding assay was performed as described in the Materials and Methods in detail using the various microbial cell wall components, β-1,3-glucan (A), LPS (B), chitin (C), cellulose (D), and peptidoglycan (E). GNBP1 (lane 1); GNBP2 (lane 2); GNBP3 (lane 3); and BSA as a negative control (lane 4).

Figure 6.

The phosphatase activity assay of the recovered soluble PTPRS (A) and DUSP7 (B) from IBs (1 mg/mL) by HHP (200 MPa, 25°C, 24 h) in the refolding buffer containing the additives. The enzymatic activity was monitored by the p-NPP hydrolysis assay as described in detail in the Materials and Methods section. The symbols represent the following buffer conditions: (○) buffer; (•) 1 M urea; (□) 2 M urea; (▪) 2.5 M glycerol; (▴) 0.5 M arginine; (▵) 0.1 mM Tween 20; (▿) 0.5 mM Triton X-100. The error bars represent the standard deviation for triplicate incubated samples.

Figure 7.

Effects of pressure (A) and duration of pressurization (B) on the solubilization yield of IBs in the refolding buffer containing 0.5 M arginine. In A, IBs (1 mg/mL) were incubated under the designated HHP at 25°C for 24 h. In B, IBs (1 mg/mL) were incubated under 200 MPa at 25°C for 2, 24, and 72 h. The symbols represent each protein as follows: GNBP1 (○); GNBP2 (□); GNBP3 (▵), PTPRS (▿), and DUSP7 (⋄). The error bars represent the standard deviation for triplicate incubated samples.

Figure 8.

Atmospheric urea solubility of IBs (A) and the plotting of the midpoint of urea solubilization versus the solubilization yields of IBs (1 mg/mL) by HHP (200 MPa, 25°C, 24 h) (B). In A, the symbols represent each protein as follows: GNBP1 (○); GNBP2 (▪); GNBP3 (▵), PTPRS (▾), and DUSP7 (⋄). In B, closed (•) and open (○) symbols represent the solubilization yields of IBs incubated in the refolding buffer alone and containing 0.5 M arginine, respectively. The error bars represent the standard deviation for triplicate incubated samples.