Chaperokine function of recombinant Hsp72 produced in insect cells using a baculovirus expression system is retained

Abstract

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72(bv) (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72(bv) enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72(bv) in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72(bv) can now be used to unlock the important role Hsp72 plays in modulating immune function.

FIGURE 1.

Schematic representation of the pBACgus-70 transfer vector construct. The coding sequence of the human hsp72 gene was cloned into baculovirus transfer plasmid pBACgus-2cp between HindIII and XhoI restriction sites to form the pBACgus-70 transfer vector. This pBACgus-70 transfer vector was cotransfected into Sf9 insect cells along with the baculovirus A. californica multiple nuclear polyhedrosis virus linear DNA to form recombinant baculovirus containing the hsp72 gene after the polyhedron (polh) gene promoter in its genome. kb, kilobase; ORF, open reading frame.

FIGURE 2.

Identification of recombinant baculovirus containing target gene. Sf9 insect cells were cotransfected with A. californica multiple nuclear polyhedrosis virus linear DNA and baculovirus transfer vectors pBACgus-70. A, cotransfected Sf9 insect cells were overlaid with SeaPlaque agarose and grown for 5 days. 5-bromo-4-chloro-3-indolyl-b-d-glucuronide (50 μl) was spread on each plate. After 3–24 h, blue plaque identified the cells containing potential recombinant virus. The recombinant virus was purified by three rounds of plaque assay. B, recombinant viral genome was extracted and verified by PCR using EcoRV forward and DOWN1629 reverse primers as described under “Experimental Procedures.” Lane 1, 1-kb DNA ladder; lane 2, pBACgus-2cp transfer vector as negative control; lane 3, pBACgus-70 transfer vector as positive control; lanes 4-18, different viral DNA extracted from independent recombinant viral isolates. The arrow indicated the recombinant baculovirus containing the hsp72 gene within its genome. The results were representative experiments from at least 10 independently performed experiments with similar results.

FIGURE 3.

Expression of recombinant human Hsp72^bv in Sf9 insect cells. A, Sf9 insect cells (2 × 10⁶ cells/ml) were infected with recombinant baculovirus virus containing the hsp72 gene. Samples were collected every 24 h post-infection and examined for the expression of Hsp72 by Western blot analysis. Briefly, membranes were probed with mouse anti-Hsp72 monoclonal antibody (top panel) or anti-GAPDH (loading control; bottom panel) followed by incubation with peroxidase-conjugated goat anti-mouse IgG secondary antibody. Lane 1, 0 h; lane 2, 24 h; lane 3, 48 h, lane 4, 72 h; lane 5, 96 h; and lane 6, 120 h. In a separate experiment 96 h post-infection, cells were collected, and clear cell lysate was applied to a Ni-NTA His·Bind resin column. Purified protein Hsp72^bv was collected and desalted using Centricon Ultracel YM-50. Hsp72^bv protein was analyzed using SDS-PAGE followed by either Coomassie blue staining (B); lane 1, protein maker; lane 2, 30 μg Hsp72^bv; lane 3, 1.5 μg Hsp72^bv or Silver staining (C); lane 1, 50 ng Hsp72^bv; lane 2, 100 ng Hsp72^bv; lane 3, 200 ng Hsp72^bv; lane 4, 500 ng Hsp72^bv. The data is a representative experiment from three independently performed experiments with similar results.

FIGURE 4.

Recombinant human Hsp72^bv expressed in Sf9 insect cells is endotoxin-free. Recombinant human Hsp72^bv protein was expressed by BEVS in Sf9 cells. Purified protein was examined for endotoxin contamination using a Limulus amebocyte lysate QCL-1000 kit (Cambrex). Briefly, the determination includes a blank for endotoxin standards in quadruplicate. Blank wells contained 50 μl of Limulus amebocyte lysate reagent water instead of the sample. Absorbance was measured at 405–410 nm. Data are the standard curve using the formula: y = 18.923x + 0.2472 (top panel). The mean absorbance (405–410 nm) and corresponding concentrations of the four standards (endotoxin) ranging from 0.01 to 0.1 ng/ml and recombinant human Hsp72^bv protein (bottom panel).

FIGURE 5.

Exogenously added recombinant human Hsp72^bv protects neuroblastoma cells against lethal heat stress. Neuroblastoma SH-SY5Y cells (10⁶/ml) were incubated in the presence of recombinant human Hsp72^bv protein (25 μg/ml; gray bars) or recombinant human Hsp72^bv protein 50 μg/ml; filled bars) or 50 μg/ml BSA (control protein; open bars) for 3 h at 37 °C. Cells were then exposed to heat shock (HS; 44 °C for 40 min), and incubated for a further 24 h at 37 °C, and cell death was measured by trypan exclusion assay. Data represent the percentage of dead cells ± S.E. and are the sum of three independently performed experiments. *, p < 0.05 versus control (BSA).

FIGURE 6.

Recombinant human Hsp72^bv induces rapid intracellular calcium flux. THP-1 cells were treated with 3 μm fluo-3 and 9 μm Fura Red cell loading medium as described in detail under “Experimental Procedures.” Briefly, after incubating at 37 °C for 30 min, cells were spun down and washed with 6 ml wash buffer to make 1 × 10⁷cells/ml cell suspension. Samples were warmed up at 37 °C for 5 min and loaded to a BD FACSAria flow cytometer for analysis. Baseline values were initially recorded for 1 min before the addition of 5 μg Hsp72^bv (green line), or 15 μg Hsp72^bv (blue line), or 25 μg Hsp72^bv (brown line), or 50 μg Hsp72^bv (purple line), or 10 μm BAPTA + 5 μg Hsp72^bv (red line). Data were plotted as fluo-3 fluorescence versus time and Fura Red fluorescence versus time. FlowJo software was used to analyze the fluo-3/Fura Red ratio versus time. The result is a representative experiment from three independently performed experiments with similar results.

FIGURE 7.

Effect of recombinant human Hsp72^bv on splenocyte functions. Mouse splenocytes (10⁶ cells) were treated with BSA (100 μg) or recombinant Hsp72^bv (100 or 200 μg) for 96 h in a 37 °C incubator. A, supernatant was recovered, and IL-4, tumor necrosis factor-α, IL-12p70, or IFN-γ was measured using cytometric bead assay according to the manufacturer's instructions (BD Biosciences) on a BD FACSAria flow cytometer. Raw data were analyzed using FCAP array software. Data are mean fluorescence intensity ± S.D. and are the sum of three independently performed experiments. *, p < 0.05 versus control (BSA). B, splenocytes were collected and stained with anti-mouse CD11-phycoerythrin, CD4-fluorescein isothiocyanate, and CD8-phycoerythrin and analyzed using a BD FACSAria flow cytometer. Individual cells were gated on the basis of forward and orthogonal scatter. The photomultiplier for fluorescein isothiocyanate (FL1-height) or PE (FL2-height) was set on a logarithmic scale. Cell debris was excluded by raising the forward scatter-height photomultiplier tube threshold. The flow rate was adjusted to <200 cells/second, and at least 30,000 cells were analyzed for each sample. Data are the sum of four independently performed experiments. *, p < 0.05 versus control (BSA).