Heterologous expression of human costimulatory molecule B7-2 and construction of B7-2 immobilized polyhydroxyalkanoate nanoparticles for use as an immune activation agent

Abstract

Background: Costimulation of T cells via costimulatory molecules such as B7 is important for eliciting cell-mediated antitumor immunity. Presenting costimulation molecules by immobilizing recombinant B7 on the surface of nanovectors is a novel strategy for complementary therapy. Polyhydroxyalkanoates (PHAs) are a family of biodegradable, non-toxic, biocompatible polyesters, which can be used as a nonspecific immobilizing matrix for protein presentation. Recombinant protein fusion with PHA granule binding protein phasin (PhaP) can be easily immobilized on the surface of PHA nanoparticles through hydrophobic interactions between PhaP and PHA, and therefore provides a low-cost protein presenting strategy.

Results: In this study, the extracellular domain of the B7-2 molecule (also named as CD86) was fused with PhaP at its N-terminal and heterogeneously expressed in recombinant Escherichia coli strain BL21 (DE3). The purified B7-2-PhaP protein was immobilized on the surface of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx)-based nanoparticles. Loading of 240 μg (3.2 pMol) of B7-2-PhaP protein per mg nanoparticles was achieved. Immobilized B7-2-PhaP on PHBHHx nanoparticles induced T cell activation and proliferation in vitro.

Conclusions: A PHA nanoparticle-based B7-2 costimulation molecule-presenting system was constructed. The PHA-based B7 presenting nanosystem provided costimulation signals to induce T cell activation and expansion in vitro. The B7-2-PhaP immobilized PHA nanosystem is a novel strategy for costimulation molecule presentation and may be used for costimulatory molecule complementary therapy.

Figure 1

Coomassie blue-stained SDS-PAGE of hetero-expressed GST-B7-2-PhaP fusion protein by recombinant E. coli BL21 (DE3) (pGEX-B72P). Lane 1: whole crude lysate of E. coli BL21 (DE3) transformed with pGEX-4T-3 plasmid. Lane 2: whole crude lysate of E. coli BL21 (DE3) transformed with pGEX-B72P plasmid. Protein weight marker (Institute of Biochemistry and Cell Biology, SIBS, CAS, China) was loaded in lane M.

Figure 2

Coomassie blue-stained SDS-PAGE of soluble GST-B7-2-PhaP fusion protein. Lane 1: the soluble fraction was recovered after centrifugation of whole crude lysate of E. coli BL21 (DE3) transformed with pGEX-B72P plasmid. Lane 2: purified soluble GST-B7-2-PhaP fusion protein. Protein weight marker (Institute of Biochemistry and Cell Biology, SIBS, CAS, China) was loaded in lane M.

Figure 3

Size distribution of PHBHHx nanoparticles determined by dynamic light scattering.

Figure 4

Western blot analysis of GST-B7-2-PhaP fusion protein immobilized on the surface of PHBHHx nanoparticles. Lanes 1-2: the original GST-B7-2-PhaP protein samples with an initial concentration of 2 and 2.5 mg ml^-1, respectively. Lanes 3-4: GST-B7-2-PhaP protein eluted from the surface of PHBHHx nanoparticles after the immobilization procedure. Lanes 5-6: the remnant GST-B7-2-PhaP proteins remaining in the supernatant after the immobilization procedure. The concentration of the initial protein sample of Lanes 3 and 5 was 2 mg ml^-1, and that of Lanes 4 and 6 was 2.5 mg ml^-1. Protein weight marker (Institute of Biochemistry and Cell Biology, SIBS, CAS, China) was loaded in lane M, which was not detected by western blot analysis.

Figure 5

Relative proliferation ratio of human peripheral blood lymphocytes by B7-2-PhaP immobilized PHBHHx nanoparticle treatment. Lymphocytes were seeded in triplicate at 1 × 10⁴ cells per well in 96-well culture plates, followed by incubation with the indicated treatments for 72 h. 1: control cells; 2: cells treated with 5 μg ml^-1 phytohemagglutinin; 3: cells treated with 500 ng ml^-1 anti-CD3 antibody; 4: cells treated with 5 μg ml^-1 PHBHHx nanoparticles; 5: cells treated with 1 μg ml^-1 B7-2-PhaP fusion protein loaded PHBHHx nanoparticles; 6: cells treated with 1 μg ml^-1 B7-2-PhaP fusion protein loaded PHBHHx nanoparticles plus 500 ng ml^-1 anti-CD3 antibody; 7: cells treated with 1 μg ml^-1 non-immobilized B7-2-PhaP fusion protein; 8: cells treated with 1 μg ml^-1 non-immobilized B7-2-PhaP fusion protein plus 500 ng ml^-1 anti-CD3 antibody. The cell proliferation was analyzed by MTT assay and the relative proliferation ratio was calculated relative to the cell proliferation level of the control group, which was defined as 100%.

Figure 6

Plasmid map of pGEX-B72-P. pGEX-B72-P was derived from plasmid pGEX-4T-3 which contains the pBR322 origin. B7-2: coding sequence for 224-aa extracellular domain of human B7-2 costimulatory molecule; phaP: coding sequence of PHA associated protein phasin (PhaP); Amp: ampicillin resistance gene; Lac I: coding sequence of Lac operon DNA-binding transcriptional repressor.