Pgex 4t2 plasmid

GRAF1b GAP-SH3 (amino acids 370–759 of hGRAF1b) and GRAF2 SH3 (amino acids 718–786 of hGRAF2) were cloned in a modified Gateway-compatible pGex 4T2 plasmid (GE Healthcare Life Sciences, Piscataway, NJ). Proteins were expressed in BL21(DE3) pLysS E. coli cells (16 h, 18°C). Cells were lysed in 20 mM HEPES pH 7.4, 500 mM NaCl, 1 mM DTT, Complete cocktail of protease inhibitors (Roche, Penzberg, Germany) by freezing–thawing and sonication. Proteins were purified on glutathione–Sepharose beads, cleaved with thrombin, and further purified by Superdex 75 (GRAF1b GAP-SH3) or Superdex 200 (GRAF2 SH3) gel filtration (GE Healthcare Life Sciences). Proteins were injected into rabbits (Eurogentec, Seraing, Belgium). Final bleed sera were purified by affinity chromatography using HiTrap NHS- activated HP columns (GE Healthcare Life Sciences). Specificity was checked using purified proteins and GRAF1^+/+ and GRAF1^−/− brain extracts.

The cell-permeable trans-activator of transcription peptide (TAT)-GILZ fusion protein and the respective control protein (TAT-Co) were generated and purified as previously (13 (link), 31 (link)). In brief, TAT and TAT-GILZ sequences were inserted into the pGEX-4T2 plasmid (GE Healthcare, #28-9545-50) to produce an in-frame fusion protein. The GST fusion protein expression was induced in E. coli BL21 (GE Healthcare, #27-1542-01) with 0.1 mM isopropyl β-d-thiogalactopyranoside (Sigma-Aldrich, #I5502). After lysis by sonication, proteins were purified with glutathione-sepharose 4B beads (GE Healthcare, #17-0756-01) according to the manufacturer's instructions. Protein purity was evaluated by SDS-PAGE and Coomassie blue staining.

Full-length wild-type SV40 VP1 and its derivative mutants were constructed with the pGEX-4T2 plasmid (GE Healthcare, Piscataway, NJ), expressed in Escherichia coli BL21(DE3) cells (Agilent) and purified by using glutathione S-transferase (GST) affinity purification and thrombin cleavage, as previously described (21 (link)). Mutant VP1 unable to form VLPs was truncated after amino acid position 305 (21 (link)).
For electron microscopy, purified pentamers were diluted in pH 7.2 PBS at 20 µg/ml and analyzed after negative staining with 1% uranyl acetate (21 (link)) via Tecnai T12 transmission electron microscopy at the Yale Electron Microscopy Core Facility. For transmission electron microscopy of infected cells, 2 × 10⁶ CV-1 cells were mock infected or infected with wild-type or A70L SV40 at an MOI of 1.5. At 64 h postinfection, samples were fixed, stained with 2% uranyl acetate, dehydrated, embedded, sectioned, and mounted onto copper grids as described elsewhere (40 (link)).
Purified pentamers were added to CV-1 cells maintained in DMEM containing 1% (vol/vol) FBS.