Journal: Scientific Reports
Article Title: SplitCore: An exceptionally versatile viral nanoparticle for native whole protein display regardless of 3D structure
Figure Lengend Snippet: SplitCore-SplitGFP-GB1: Triple-layer fluorescent particles with exposed targeting domains. (a) Design of SplitCore-splitGFP-GB1 fusions (SCSGFP-GB1). GB1 fused to one of the new ends provided by the split GFP insert (see Supplementary Fig. S3 online) should yield fluorescent CLPs with exposed immunoglobulin binding GB1 domains. (b) Expression and CLP formation. Bacterial lysates were subjected to sedimentation as in Fig. 1c . SDS-PAGE (top) demonstrated cosedimentation of two proteins of the expected sizes into fractions 8 to 12 which produced distinct green fluorescent bands in NAGE (UV) that also stained with CB. Similar results were obtained with GB1 fused to GFPβ1-10 on coreN (not shown). (c) Visualization of folded GFP containing material in the gradient tube from which the samples in (b) were derived. (d) Negative staining EM. The two micrographs are from independent grids onto which material from fraction 8, diluted 10-fold in PBS, was applied. Smaller (*; diameter ∼38 nm) and larger (**; diameter ∼42 nm) particles were observed. (e,f) SCSGFP-GB1 binds antibodies. (e) Indirect immunofluorescence. Permeabilized HeLa cells were incubated with mouse anti-tubulin antibody (i, iii) or not (ii, iv), then with SCSGFP-GB1 (i, ii) or an Alexa488-conjugated goat anti-mouse IgG (gαm-Alexa488) antibody (iii, iv). Both generated similar, primary antibody-dependent staining patterns. (f) Fluorescent Western blot. Decreasing amounts of GFP were separated by SDS-PAGE, blotted on nitrocellulose and incubated with a mouse anti-GFP mAb mixture (Roche), then with either gαm-Alexa488 or SC-SGFP-GB1. Fluorescent signals were detected using a Typhoon 7000 imager (GE Healthcare).
Article Snippet: Immunoglobulin binding by SC-SGFP-GB1 CLPs For indirect immunofluorescence, 4% paraformaldehyde fixed HeLa cells were permeabilized using 0.2 % Triton X-100 in PBS, blocked with 5% BSA in PBS and incubated with 0.05 µg/ml of mouse-anti-tubulin mAb, or not; for detection, either 0.13 µM goat-anti-mouse IgG antibody conjugated to Alexa488 (gαm-Alexa488; Invitrogen) or 0.05 µM SC-SGFP-GB1 CLPs were used.
Techniques: Binding Assay, Expressing, Sedimentation, SDS Page, Produced, Staining, Derivative Assay, Negative Staining, Immunofluorescence, Incubation, Generated, Western Blot