An improved genetic system for bioengineering buoyant gas vesicle nanoparticles from Haloarchaea

BMC Biotechnology

Shiladitya DasSarma²  Ram Karan²  Priya DasSarma²  Susan Barnes²  Folasade Ekulona²  Barbara Smith¹ 

[1] Johns Hopkins School of Medicine Microscope Facility, Baltimore, MD 21205, USA

[2] Institute of Marine and Environmental Technology and Department of Microbiology and Immunology, University of Maryland School of Medicine, 701 E Pratt Street, Baltimore, MD 21202, USA

关键词: Luciferase;    Archaea;    Halophiles;    Vaccine;   

Others  :  835005
DOI  :  10.1186/1472-6750-13-112

 received in 2013-11-04, accepted in 2013-12-17,  发布年份 2013

【 摘 要 】

Background

Gas vesicles are hollow, buoyant organelles bounded by a thin and extremely stable protein membrane. They are coded by a cluster of gvp genes in the halophilic archaeon, Halobacterium sp. NRC-1. Using an expression vector containing the entire gvp gene cluster, gas vesicle nanoparticles (GVNPs) have been successfully bioengineered for antigen display by constructing gene fusions between the gvpC gene and coding sequences from bacterial and viral pathogens.

Results

To improve and streamline the genetic system for bioengineering of GVNPs, we first constructed a strain of Halobacterium sp. NRC-1 deleted solely for the gvpC gene. The deleted strain contained smaller, more spindle-shaped nanoparticles observable by transmission electron microscopy, confirming a shape-determining role for GvpC in gas vesicle biogenesis. Next, we constructed expression plasmids containing N-terminal coding portions or the complete gvpC gene. After introducing the expression plasmids into the Halobacterium sp. NRC-1 ΔgvpC strain, GvpC protein and variants were localized to the GVNPs by Western blotting analysis and their effects on increasing the size and shape of nanoparticles established by electron microscopy. Finally, a synthetic gene coding for Gaussia princeps luciferase was fused to the gvpC gene fragments on expression plasmids, resulting in an enzymatically active GvpC-luciferase fusion protein bound to the buoyant nanoparticles from Halobacterium.

Conclusion

GvpC protein and its N-terminal fragments expressed from plasmid constructs complemented a Halobacterium sp. NRC-1 ΔgvpC strain and bound to buoyant GVNPs. Fusion of the luciferase reporter gene from Gaussia princeps to the gvpC gene derivatives in expression plasmids produced GVNPs with enzymatically active luciferase bound. These results establish a significantly improved genetic system for displaying foreign proteins on Halobacterium gas vesicles and extend the bioengineering potential of these novel nanoparticles to catalytically active enzymes.

【 授权许可】

   
2013 DasSarma et al.; licensee BioMed Central Ltd.

【 图 表 】

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