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

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. Video LinkThe video component of this article can be found at https://www.jove.com/video/60103/ 9 . This candidate was recognized by known monoclonal antibodies to HIV-1 envelope protein. Mice immunized with V1V2-SHB-SAPN raised V1V2 specific antibodies, that, most importantly, bound to gp70 V1V2, the correct conformational epitopes 9 . The SHB-SAPN core could have other functions beyond the role as a carrier for the HIV-1 V1V2 loop. Here we describe a detailed methodology for the expression, purification, refolding, and validation of the SHB-SAPN core. The sequence selection, nanoparticle design, the molecular cloning, and transformation of E. coli have been previously described 9 . Journal of Visualized Experiments August 2019 | 150 | e60103 | Page 2 of 11 Protocol 1. Expression of the SHB-SAPN Protein in E. coli BL21(DE3) 1. Mix 95 mL of component A and 5 mL of component B of the media in a 2 L sterile glass Erlenmeyer flask as per the manufacturer's instructions (see theTable of Materials). Add ampicillin to a final concentration of 100 μg/mL. 2. Inoculate the media with E. coli from a previously established glycerol stock culture. Incubate culture at 30 °C with shaking at 200 rotations per minute (rpm) for 48 h. NOTE: The used E. coli BL21 (DE3) stock contained the ampicillin resistant expression vector 23 with the SHB-SAPN gene. Although the general protocol of the media recommends 24 h of incubation at 37 °C, 48 h of incubation at 30 °C gave higher yield for SHB-SAPN. 3. Transfer the culture to two 50 mL conical tubes. Centrifuge the tubes at 4,000 x g for 10 min with a fixed angle rotor at 4 °C. Remove the supernatant and save the pellet to harvest cells. NOTE: The cell pellet can be either processed immediately or frozen at -80 °C until use. 2. Lysis of E. coli BL21(DE3) by sonication NOTE: Use nonpyrogenic plasticware and glassware baked at 250 °C for at least 30 min. Tris(2-car...

Design and development of a self-assembling protein nanoparticle displaying PfHAP2 antigenic determinants recognized by natural acquired antibodies

“…Since the use of the universal T-helper epitopes in vaccine design is a great help in increasing immunogenicity, and due to the effectiveness of the PADRE T-helper epitope in eliciting the immune response [ 21 , 22 , 44 – 47 ], this sequence was also used in PfGCS1-SAPN design to enhance the immunogenicity, that remains to be tested in future studies. Similar to previous studies [ 21 , 22 , 35 , 43 – 46 , 48 , 49 ], the designed amino acid sequence was confirmed to form the SAPN by assembling the monomers and surface exposure of antigenic determinants as confirmed by 3D structure prediction and assembly analysis. Besides, antigenic determinants are expressed on the surface of SAPNs formed by PfGCS1-SAPNs as verified by the ELISA.…”

De NovoDesigned Peptide and Protein Hairpins Self-assemble into Sheets and Nanoparticles

“…Specifically, the proximal pin allows splaying of the helical termini, which in turn leads to curved arrays that can close, whereas the distal pins give a more tightly constrained hairpin structure, consistent with building blocks required to make flat sheets. As noted earlier, others have developed similar self-assembling peptide and protein based nanoparticles [22][23][24][25][26][27][28][29][30] or sheets, [12][13][14][15][16][17][18] so what are the differences and advantages to our hairpin system? First, by including two points (loop and pin) that define the relative positions and rotational freedom of the two coiled-coil components, we are able to control the topology of the self-assembled structures by design in a single system to render closed nanoparticles or extended sheets.…”

“…11 Such structures are usually computationally driven towards forming closely packed 2D arrays [12][13][14][15][16][17][18] , tubes [19][20][21] or 3D icosahedral particles. [22][23][24][25][26][27][28][29][30] However, these beautifully ordered near crystalline assemblies may not be amenable to decoration with large cargos, or be very permeable to small molecules due to their close packed nature. Also, these designed arrays can require thermal annealing to assemble, 15,16 which may reduce or even destroy the activity of any appended functional proteins.…”

“…25,28 This group have been particularly successful at developing these as vaccine platforms for the presenation of antigenic peptides. [26][27][28][29][30] In a different concept Marsh and colleagues combine de novo coiled-coil units and natural oligomeric proteins to render defined protein nanocages. [41][42][43][44] Here we present a self-assembling hairpin designs that form two distinct suporamolecular assemblies, namely, closed nanoparticles and extended sheets.…”

Toll like receptors agonists-based nanomedicines as veterinary immunotherapies

“…SAPN are agents known to provide intracellular delivery [186]. The production of SAPN at an industrial scale in GMP conditions for human vaccines, based on a system of expression in E. coli, has recently been described [187]. Curiously, in the studies of Karch et al, 2017 andLi et al, 2018, SAPN were linked to flagellin, an agonist of the non-endocytic TLR 5 located on the cell surface.…”