E coli bl21

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.

For purification of GST-fusion proteins, the GST expression vector (pGex-6P, GE Healthcare) as well as the Flag-expression vector pCMV-Tag2B, both containing the respective insert (murine PID1; wild type and mutated LRP1 ICD; see Figure S2 for structure and mutations of the intracellular LRP1 domain) were transformed into E. coli BL21 (Stratagene). GST fusion proteins were purified via Glutathion Sepharose 4B (GE Healthcare). LRP1-ICDs were released from the column by incubation with PreScission protease (GE Healthcare). Integrity of all recombinant proteins was confirmed by SDS-PAGE. For pulldown analysis either cell or tissue lysates (150 μg) were incubated over night at 4 °C with GST-fusion proteins. Subsequently pulldown and unbound fractions were separated by centrifugation for 3 min at 9000 rpm. Supernatants were harvested (unbound fraction) and sepharose pellets were washed six times using PBS. Finally, pellets were reduced, and PID1-interacting proteins were analysed by Western blotting.

The coding sequence of EF-1A was codon optimized and synthesized (General Biol Inc., China), and cloned into the pCold-II or pCold-TF vectors (TaKaRa Bio Inc., Japan). The pCold-II has a N-terminal His tag, whereas the pCold-TF contains a N-terminal His tag and a soluble trigger factor chaperone tag (TF) that contributes the solution of target protein. These resulting recombinant vectors were then transformed into Escherichia coli BL21 (TaKaRa Bio Inc., Japan) for protein expression. Briefly, the E. coli BL21 that bears the recombinant vector were inoculated in Luria-Bertani liquid medium containing 100 μg/mL, and incubated at 37 °C until the OD₆₀₀ reached 0.6, and were induced with 0.2 mM isopropyl β-D-1-thiogalactopyranodside at 15 °C for 16–18 h. The target EF-1A protein was purified by HisTrap HP column in ÄKATA Pure Protein Purification System (GE Healthcare Inc., USA). For protein that was expressed in pCold-TF vector, the TF tag was removed by HRV 3 C Protease (TaKaRa Bio Inc., Japan). All the purified EF-1A proteins were concerned by 10 K AIMCO Ultra-15 (Millipore Inc., USA), and were determined by Bradford Protein Assay Kit (Byotime Bio Inc., China).

Expression of E. coli invasin and H. pylori CagY MRR was detected by electrophoresis of lysates of liquid-cultured bacteria as described previously (21 (link)), using polyclonal rabbit antisera to invasin (1:15,000) or CagY MRR (1:10,000) as the primary antibodies. Detection of CagY expression in the ΔcagY_MRR mutant, which contains an in-frame deletion of the MRR, was performed using antiserum from rabbits immunized with the VirB10 portion at the C terminus of CagY (1:1,000). To generate the antiserum, DNA encoding the C terminus of H. pylori J166 CagY was PCR amplified (Table S1), cloned into pGEX-4T-3 vector, and transformed into E. coli BL21 (both from GE Healthcare). Expression of the glutathione S-transferase (GST)-fusion protein and preparation of cell extracts were performed according to the manufacturer’s instructions. The GST-fusion protein was bound to glutathione Sepharose 4B (GE Healthcare) in a column, and the GST was cleaved off by thrombin. The eluate was run on SDS-PAGE, and the purified CagY C-terminus protein was cut out from the gel and used to generate rabbit antisera according to standard protocols (Antibodies, Inc., Davis, CA).