Figure 1.
SDS-PAGE analysis of expression and purification of rhKGF-1.
Panel A. SDS-PAGE analysis of the fractions collected from Glutathione Sepharose chromatography. Lane M, molecular weight standards; lane 1, the cell lysate supernatant of BL21 (DE3) containing pET- GST-KGF-1 induced with 1.0 mM IPTG for 4 h; lane 2, the flow-through fraction; lane 3, the fraction eluted with 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0. Panel B. SDS-PAGE analysis of the fractions collected from CM Sepharose chromatography. Lane M, molecular weight standards; lane 4, the GST-rhKGF-1 fusion protein after treatment with thrombin; lane 5, the flow-through fraction; lane 6, NaCl-eluted fraction. Panel C. SDS-PAGE analysis of Heparin Sepharose chromatography. Lane M, molecular weight standards; lane 7, NaCl-eluted fraction from CM Sepharose Fast Flow column; lane 8, 0.45 M NaCl-eluted fraction; lane 8, 0.6 M NaCl-eluted fraction.
Figure 2.
SDS-PAGE analysis and elution profile of solid-phase PEGylation reaction mixture.
Panel A. SDS-PAGE analysis of solid-phase PEGylated rhKGF-1. Lane M, molecular weight standards; lane a, solid-phase PEGylation products obtained at PEG-to-protein ratio of 10/1 and reaction time of 8 h; lane b, non-PEGylated rhKGF-1. Panel B. Elution profile of PEGylation reaction mixture on a SP Sepharose Fast Flow column. The Insert panel shows SDS-PAGE analysis of the fractions collected from SP Sepharose chromatography.
Figure 3.
Validation of the solid-phase PEGylated rhKGF-1.
Panel A, MALDI-TOF mass spectrometry of PEGylated rhKGF-1 showing the molecular mass of PEGylated rhKGF-1 (36997 Da). Panel B, MALDI-TOF mass spectrometry of non-PEGylated rhKGF-1 showing the molecular mass of non-PEGylated rhKGF-1 (16297 Da). Panel C, D. N-terminal sequencing of non-PEGylated and PEGylated rhKGF-1 by Edman degradation method.
Figure 4.
The effect of rhKGF-1 PEGylation on MAP kinase activation.
Panel A. NIH 3T3 fibroblasts were stimulated with rhKGF-1 or PEGylated rhKGF-1 at three does 1.5 µM, 3 µM, and 6 µM, and phosphorylation courses of ERK1/2 (p-ERK) were measured by immunoblotting alongside total ERK as loading controls. The blots shown are representative of three independent experiments; Panel B. Semi-quantitative analysis of the protein bands in Panel A. *, p<0.05 vs Normal control; #, p<0.05 vs the corresponding non-PEGylated rhKGF-1 group.
Table 1.
Biological activity of native rhKGF-1 and PEGylated rhKGF-1.
Figure 5.
The effect of solid-phase PEGylation on the structural integrity and stability of rhKGF-1.
Panel A. Far-UV CD spectra of non-PEGylated (black line) and solid-phase PEGylated rhKGF-1 (red line). The ellipticities are reported as mean residue ellipticity (MRE) (deg cm2 dmol−1). Panel B. Effect of PEGyaltion on the proteolytic stability of rhKGF-1. PEGylated and intact rhKGF-1 were incubated with trypsin with a molar ratio of 10/1 (protein/trypsin) at 37°C for the indicated periods of times. Trypsin-treated rhKGF-1 was examined for the protein integrity by SDS-PAGE. The bands representing PEGylated and intact rhKGF-1 were quantified by densitometry scanning. The band densities of non-trypsin-treated intact and PEGylated rhKGF-1 are considered as 100% (indicated as control), while the band densities of trypsin-treated PEGylated or intact rhKGF-1 are presented as relative percentage to the control. *, p<0.05 vs the corresponding intact rhKGF-1 group. Panel C. Kinetic plots of remaining soluble non-PEGylated rhKGF-1 (black square) and PEGylated rhKGF-1 (red circle). The soluble protein concentration was determined by Bicinchoninic acid (BCA) kit. Samples were first incubated at 37°C for 3 days and then transferred to 45°C for the rest of the experiments. Pane D. Effect of pH on in vitro bioactivity of non-PEGylated rhKGF-1 (black square) and PEGylated rhKGF-1 (red circle).
Figure 6.
Pharmacokinetics study of solid-phase PEGylated rhKGF-1 in rats.
Panel A. Normal male SD rats were injected intravenously with 100 µg/kg rhKGF-1 (open circle), and PEGylated rhKGF-1 (solid circle). Blood samples were collected at the indicated time points. The amount of rhKGF-1 was measured by Human KGF-basic Mini ELISA Development Kit. A standard curve was made for each rhKGF-1, n = 6. Values are the mean±SD. Panel B. Comparison of half-life of non-PEGylated rhKGF-1 and PEGylated rhKGF-1. *, p<0.01 vs the corresponding non-PEGylated rhKGF-1 group.
Figure 7.
Effect of solid-phase PEGylated rhKGF-1 on serum AST and ALT levels in rats intoxicated with CCl4.
48 male SD rats were randomly divided into four groups (12 rats/group): enalpril interventional group A (rhKGF-1-pretreated groups), enalpril interventional group B (PEGylated rhKGF-1-pretreated groups), injury-model group and healthy control group. The test groups were pretreated with non-PEGylated and PEGylated rhKGF-1 24 hours prior to 2.5 mL/kg CCl4 administration. Four hours after the intraperitoneal injection of CCl4, rats were killed and their serum AST/ALT levels were determined. *, p<0.05 vs control group; #, p<0.05 vs injury-model group; ss, p<0.05 between indicated groups.