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Abstract

Bioengineering

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Published: August 21st, 2019

DOI:

10.3791/60103

1U.S. Military HIV Research Program, Walter Reed Army Institute of Research, 2Henry M. Jackson Foundation for the Advancement of Military Medicine, 3Alpha-O Peptides AG

Abstract

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

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Keywords E Coli

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