bira plasmid (pdisplay-bira-er)  (Addgene inc)

Journal: Structure (London, England : 1993)

Article Title: Structural insight into rabies virus neutralization revealed by an engineered antibody scaffold.

doi: 10.1016/j.str.2024.10.002

Figure Lengend Snippet: Figure 2. Crystal structure of the CR57 diabody in complex with RABV-G domain III (A) Domain schematic of RABV-G shows the organization of the glycoprotein, including the signal peptide (SP), domains I–IV (colored red, blue, orange, and yellow, respectively), ectodomain C-terminus (gray), transmembrane domain (TM), and intraviral domain (black). The schematic was produced using the DOG software.37 Composition of the domain III construct used in this study is presented alongside an illustration of domain III organization in the context of the trimeric prefusion RABV-G spike (PDB: 7U9G). (B) Crystal structure of the CR57 diabody in complex with RABV-G domain III, resolved at 2.70 A˚ resolution. The lower panel includes a magnified view of the CR57 diabody-RABV-G domain III interface, where key interacting residues are labeled and shown as sticks with oxygen atoms in red and nitrogen atoms in blue.

Article Snippet: Protein preparation for Biolayer interferometry The wild-type RABV-G domain III construct encoding residues 32–59 and 181–259 (GenBank: AAC34683.1) connected by a linker composed of three glycines, with a C-terminal Avitag (sequence: GLNDIFEAQKIEWHE) before C-terminal 63 His tag, and its K226E mutant, were designed and commercially obtained as codon-optimized synthetic cDNA from GeneArt (Thermo Fisher Scientific), and cloned into the pHL-sec mammalian expression vector (Addgene #99845).55 The domain III expression plasmid was transiently co-transfected with pDisplay-BirA-ER (Addgene #20856)57 plasmid for in vivo biotinylation of the Avitag.

Techniques: Produced, Software, Construct, Labeling

Journal: Structure (London, England : 1993)

Article Title: Structural insight into rabies virus neutralization revealed by an engineered antibody scaffold.

doi: 10.1016/j.str.2024.10.002

Figure Lengend Snippet: Figure 3. Comparative analysis of neutralizing antibody epitopes on the RABV-G spike (A) Superposition of CR57 diabody-RABV-G domain III complex structure (for clarity, only a single CR57 Fv is shown) with a previously reported prefusion RABV-G spike (PDB: 7U9G15). (B) Surface representation of the spike (side and top views), displaying the epitope footprints of antibodies targeting domain III, which comprise CR57 (delineated in cyan), RVC20 (dark green), and 1112-1 (light green), and epitopes of antibodies targeting domains I and II, which comprise 523-11 (white), 17C7 (purple), and RVA122 (pink). (C) A close-up of the key interacting region between domain III and mAbs CR57; the same region is also shown for mAbs RVC20 and 1112-1 interfaces. RABV-G domain III is shown in orange, with the peptide KLCGVL highlighted by a dashed circle. CDRH3 is shown as a bold loop and CDRH3 residues interacting with the KLCGVL peptide are labeled and shown as sticks with oxygen atoms in red and nitrogen atoms in blue.

Article Snippet: Protein preparation for Biolayer interferometry The wild-type RABV-G domain III construct encoding residues 32–59 and 181–259 (GenBank: AAC34683.1) connected by a linker composed of three glycines, with a C-terminal Avitag (sequence: GLNDIFEAQKIEWHE) before C-terminal 63 His tag, and its K226E mutant, were designed and commercially obtained as codon-optimized synthetic cDNA from GeneArt (Thermo Fisher Scientific), and cloned into the pHL-sec mammalian expression vector (Addgene #99845).55 The domain III expression plasmid was transiently co-transfected with pDisplay-BirA-ER (Addgene #20856)57 plasmid for in vivo biotinylation of the Avitag.

Techniques: Labeling

Journal: Structure (London, England : 1993)

Article Title: Structural insight into rabies virus neutralization revealed by an engineered antibody scaffold.

doi: 10.1016/j.str.2024.10.002

Figure Lengend Snippet: Figure 4. CR57 binding overlaps with a putative nAChR-interacting region and abrogates spike-mediated fusion (A) Superposition of CR57RABV-G domain III complex onto the structure of the trimeric prefusion spike (PDB: 7U9G,15 for clarity only a single CR57 Fv is displayed). (A) and (B) illustrate the occlusion of the nAChR-binding RABV-G peptide by CR57 Fv. The nAChR binding region is composed of RABV-G residues 175–203 and is colored dark magenta, with the minimal peptide implicated for nAChR binding, residues 187–199, highlighted in magenta.

Article Snippet: Protein preparation for Biolayer interferometry The wild-type RABV-G domain III construct encoding residues 32–59 and 181–259 (GenBank: AAC34683.1) connected by a linker composed of three glycines, with a C-terminal Avitag (sequence: GLNDIFEAQKIEWHE) before C-terminal 63 His tag, and its K226E mutant, were designed and commercially obtained as codon-optimized synthetic cDNA from GeneArt (Thermo Fisher Scientific), and cloned into the pHL-sec mammalian expression vector (Addgene #99845).55 The domain III expression plasmid was transiently co-transfected with pDisplay-BirA-ER (Addgene #20856)57 plasmid for in vivo biotinylation of the Avitag.

Techniques: Binding Assay