Journal: Vaccines

Article Title: Molecular Design of Encapsulin Protein Nanoparticles to Display Rotavirus Antigens for Enhancing Immunogenicity

doi: 10.3390/vaccines12091020

Figure Lengend Snippet: Design and soluble expression of the fusion protein of rotavirus receptor-binding domain and ENC nanoparticles. ( a ) Computational modeling and theoretical structure of ENC-RV VP8*. The predicted structure of ENC and linked VP8* are colored blue and green, respectively. The fusion protein of ENC-VP8* was predicted using SWISS-MODEL, Expasy. ( b ) Schematic designs of the protein structures. The size of proteins is shown under the name of each protein. ( c ) Expression of ENC and ENC-RV VP8* in the presence or absence of the RID fusion partner. The proteins were expressed at various vector constructions, and the cell lysates were separated into T, P, and S fractions via centrifugation and analyzed using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ENC-VP8* with RID fusion partner is indicated by the red arrow. Lane M indicates molecular size markers. TEV SITE, tobacco etch virus (TEV) protease cleavage site; T, total fraction; P, insoluble pellet fraction; S, soluble fraction; ENC, encapsulin; RV, rotavirus vaccine, RID, RNA-interacting domain.

Article Snippet: MA104 cells were seeded at 1.0 × 10 4 cells/well in 96-well plates (Thermo Fisher Scientific, København V, Denmark) with 200 μL of complete Dulbecco’s modified Eagle medium (containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 1–2 d. Rotavirus Wa strain (G1P [ ]) (Rotavirus A; ATCC CRL-2378.1, Manassas, VA, USA) was thawed from −80 °C storage and activated via incubation with trypsin (final concentration of 10 μg/mL) for 1 h at 37 °C.

Techniques: Expressing, Binding Assay, Plasmid Preparation, Centrifugation, Polyacrylamide Gel Electrophoresis, Virus

Journal: Vaccines

Article Title: Molecular Design of Encapsulin Protein Nanoparticles to Display Rotavirus Antigens for Enhancing Immunogenicity

doi: 10.3390/vaccines12091020

Figure Lengend Snippet: The addition of GS linker to the N-terminal of fusion protein increases the cleavage efficiency of TEV protease. ( a ) Schematic diagram of the expression vector system. The two linkers are shown in red (GS) 3 and white (Linker) and TEV SITE indicates the TEV protease recognition site. ( b ) Time-lapse 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of TEV cleavage before (left panel) and after (right panel) the linker insertion. Lane M indicates molecular size markers. Proteins purified or separated until purification are indicated by red arrows. ( c ) Following IMAC elution, minor residual contaminants and non-target proteins were removed. Purified NPs (ENC NPs with and without VP8*) were negatively stained and analyzed via transmission electron microscopy. Scale bars = 100 nm and 200 nm. ENC, encapsulin; RV, rotavirus vaccine, RID, RNA-interacting domain; M, molecular weight marker; IMAC, elution with anion exchange chromatography.

Article Snippet: MA104 cells were seeded at 1.0 × 10 4 cells/well in 96-well plates (Thermo Fisher Scientific, København V, Denmark) with 200 μL of complete Dulbecco’s modified Eagle medium (containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 1–2 d. Rotavirus Wa strain (G1P [ ]) (Rotavirus A; ATCC CRL-2378.1, Manassas, VA, USA) was thawed from −80 °C storage and activated via incubation with trypsin (final concentration of 10 μg/mL) for 1 h at 37 °C.

Techniques: Expressing, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Purification, Staining, Transmission Assay, Electron Microscopy, Molecular Weight, Marker, Chromatography

Journal: Vaccines

Article Title: Molecular Design of Encapsulin Protein Nanoparticles to Display Rotavirus Antigens for Enhancing Immunogenicity

doi: 10.3390/vaccines12091020

Figure Lengend Snippet: Linker affects the enhanced yield of nanoparticle (NP) antigen. ( a ) Schematic diagram of ENC-RV VP8* with inserted linker between ENC and VP8* antigen. Underlined L2 is colored as white. ENC and linked VP8* are colored blue and green, respectively. ( b ) The solubility of recombinant proteins fused with different linkers was analyzed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Protein expression is under the control of isopropyl-β-D-thiogalactopyranoside induction. NP protein was fused with the C-terminal antigen VP8* via the linkers, L1: (G 3 S) 3 SGGS linker (left) and L2: (G 3 S) 2 EAAAKG 3 S linker (right). The arrow indicates the band position of each recombinant protein. ( c ) Dynamic light scattering measurement profiles (three independent scans of the same sample) of particle sizes and distributions of ENC NPs with L1 (left) and L2 (right). ( d ) Purified NPs were negatively stained and analyzed via transmission electron microscopy. Scale bar = 200 nm. M, molecular weight marker; T, total fraction; P, insoluble pellet fraction; S, soluble fraction; L1, linker 1; L2, linker 2; ENC, encapsulin; RV, rotavirus vaccine, RID, RNA-interacting domain.

Article Snippet: MA104 cells were seeded at 1.0 × 10 4 cells/well in 96-well plates (Thermo Fisher Scientific, København V, Denmark) with 200 μL of complete Dulbecco’s modified Eagle medium (containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 1–2 d. Rotavirus Wa strain (G1P [ ]) (Rotavirus A; ATCC CRL-2378.1, Manassas, VA, USA) was thawed from −80 °C storage and activated via incubation with trypsin (final concentration of 10 μg/mL) for 1 h at 37 °C.

Techniques: Solubility, Recombinant, Polyacrylamide Gel Electrophoresis, Expressing, Control, Purification, Staining, Transmission Assay, Electron Microscopy, Molecular Weight, Marker

Journal: Vaccines

Article Title: Molecular Design of Encapsulin Protein Nanoparticles to Display Rotavirus Antigens for Enhancing Immunogenicity

doi: 10.3390/vaccines12091020

Figure Lengend Snippet: Conformation of VP8* displayed with P2 on ENC was self-assembled into 60-mer nanoparticles (NPs). ( a ) Schematic diagram of ENC-RV VP8* with inserted linker between ENC and VP8* antigen. P2 is colored orange. ENC and linked VP8* are colored blue and green, respectively. ( b ) Purified recombinant proteins fused with different linkers were analyzed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. NP protein was fused with the C-terminal antigen VP8* via the L2 linker with P2 (right). The arrow indicates the band position of each recombinant protein. ( c ) Dynamic light scattering measurement profiles (three independent scans of the same sample) of particle sizes and distributions of ENC NPs with P2. ( d ) Purified NPs were negatively stained and analyzed via transmission electron microscopy. Scale bar = 50 nm. ENC, encapsulin; L2, linker 2; RV, rotavirus vaccine; RSD, relative standard deviation.

Article Snippet: MA104 cells were seeded at 1.0 × 10 4 cells/well in 96-well plates (Thermo Fisher Scientific, København V, Denmark) with 200 μL of complete Dulbecco’s modified Eagle medium (containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 1–2 d. Rotavirus Wa strain (G1P [ ]) (Rotavirus A; ATCC CRL-2378.1, Manassas, VA, USA) was thawed from −80 °C storage and activated via incubation with trypsin (final concentration of 10 μg/mL) for 1 h at 37 °C.

Techniques: Purification, Recombinant, Polyacrylamide Gel Electrophoresis, Staining, Transmission Assay, Electron Microscopy, Standard Deviation