Stability of fungal immunomodulatory protein, FIP-gts and FIP-fve, in IFN-γ production

Figures & data

Figure 1.  Effects of FIP-gts and FIP-fve on IFN-γ secretion in PBMCs. (A) Effects of FIP-gts on IFN-γ secretion in hPBMCs. Cultured hPBMCs (2×10⁶ cells/ml, 1 ml/well) were treated with the indicated concentrations of FIP-gts in RPMI 1640 supplemented with 10% FBS for 48 h. Conditioned media were subjected to ELISA to measure amounts of secreted IFN-γ (mean value from three independent experiments). (B) Effects of FIP-fve on IFN-γ secretion in hPBMCs. Cultured peripheral T cells (2×10⁶ cells/ml, 1 ml/well) were treated with the indicated concentrations of FIP-fve in RPMI 1640 supplemented with 10% FBS for 48 h. Conditioned media were subjected to ELISA to measure amounts of secreted IFN-γ (mean value from three independent experiments).

Figure 2.  Effect of heating on the stability of FIP-gts and FIP-fve-induced IFN-γ secretion in hPBMCs. The FIP-gts and FIP-fve were pre-incubated at 100°C from 0 to 8 min and residual immunomodulatory activity was determined. Cells cultured without stimulant in the culture supernatants were used as control samples. Human PBMC (2×10⁶ cells/ml) were stimulated by non-heated and heated FIP-gts (0.3 µM) and FIP-fve (7.69 µM) for 48 h. Culture supernatant was collected and assayed for IFN-γ levels by ELISA. The IFN-γ is expressed as pg/ml (mean value from three independent experiments).

Figure 3.  Stability of the deep-frozen and lyophilised FIP-fve with and without additives during storage. The freshly prepared FIP-fve was divided into five groups: (1) only lyophilised; (2) lyophilised with 30% sucrose; (3) lyophilised with 3.2M ammonium sulphate; (4) deep-frozen; and (5) deep-frozen with glycerol. The production of IFN-γ was measured by ELISA. Three independent experiments were performed. Values are means of three independent experiments.

Figure 4.  SDS-PAGE analysis of the degradation of FIP-gts and FIP-fve in SGF. (A) FIP-gts and (B) FIP-fve were treated with SGF. Molecular weight markers (lane M) are indicated on the left-hand side of the gel. SGF and the test protein control were run along. The numbers on top denote the incubation times in minutes. The FIP-gts (A) and FIP-fve (B) in SGF digestion were labelled at the right-hand side of the gel, respectively.

Figure 5.  SDS-PAGE analysis of the degradation of FIP-gts and FIP-fve in SIF. (A) FIP-gts and (B) FIP-fve were treated with SIF. Molecular weight markers (lane M) are indicated on the left-hand side of the gel. SIF and the test protein control were run along. The numbers on top denote the incubation times in minutes. The FIP-gts (A) and FIP-fve (B) in SIF digestion were labelled at the right-hand side of the gel.

Figure 6.  Chemical cross-linking of FIP-gts and FIP-fve. The dimerisation of FIP-gts and FIP-fve were examined by glutaraldehyde cross-linking reaction. About 12 µg of protein was loaded in each lane, and gels were stained with Coomassie blue.

Table 1. The IFN-γ production in dimerisation of FIP-gts and FIP-fve.

Stimulus	Glutaraldehyde (250 µM)	IFN-γ (pg/ml)
Unreated	−	<16
	+	ND
FIP-gts 0.3 (µM)	−	1021±36
	+	1201±57
FIP-fve 7.69 (µM)	−	1220±45
	+	138±51
Note: The dimerisation of FIP-gts and FIP-fve were examined by glutaraldehyde cross-linking reaction. FIP-gts (0.3 µM) and FIP-fve (7.69 µM) were treated with or without 250 µM glutaraldehyde, and further incubated with hPBMCs for 48 h. Culture supernatant was assayed by ELISA, and the results are expressed as pg/ml (mean value from three independent experiments). ND – not determined.