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

Ming-Chuan Li,Qian-Qian Liu,Ya-Li Zhang,Lei-Lei Wang,Xiao-Yun Lu

doi:10.1186/1472-6750-12-43

Abstract

BackgroundCostimulation 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.ResultsIn 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.ConclusionsA 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.

Highlights

Costimulation of T cells via costimulatory molecules such as B7 is important for eliciting cell-mediated antitumor immunity
Microbial production and purification of GST-B7-2-PhaP fusion proteins by Escherichia coli The PhaP gene was fused with the coding sequence for the extracellular domain of the B7-2 molecule and was expressed in Escherichia coli BL21 (DE3), with a glutathione S-transferase (GST) tag at the N-terminal of the resulting fusion protein
Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) analysis showed that soluble GST-B7-2-PhaP fusion protein with a molecular mass of 73 kDa was successfully produced in the recombinant E. coli BL21 (DE3) and a single band of the purified GST-B7-2-PhaP fusion protein was detected from the eluted sample (Figure 2)

Summary

Introduction

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. Recombinant protein fusion with PHA granule binding protein phasin (PhaP) can be immobilized on the surface of PHA nanoparticles through hydrophobic interactions between PhaP and PHA, and provides a low-cost protein presenting strategy. Expression of B7 on tumor cells induced tumor rejection in a murine model, suggesting that providing extra costimulation molecules might render tumor cells capable of effective antigen presentation, leading to their eradication in vivo. Based on the safety considerations of expressing B7 using a virus vector, we propose a novel strategy to present costimulatory molecules by immobilizing recombinant B7 on the surface of polyhydroxyalkanoate (PHAs)-based nanoparticles

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Discussion

Conclusion