Pgex 4t 2 vector

For GST-binding assays, full-length SPARC, SPARC N-terminal (aa 18–134) and SPARC C-terminal (aa 154–303), SPARC aa 154–175, SPARC aa 176–256, SPARC aa 257–303, SPARC aa 154–256, SPARC aa 176–303, and SPARC aa 18–266 constructs were subcloned into the pGEX4T2 vector (GE Healthcare). All GST-fusion proteins were prepared and purified from Rosetta BL21 DE3 E. coli using Glutathione sepharose 4B beads (GE Healthcare). The pET27 TGFBI vector was a kind gift from Dr. Ching Yuan (University of Minnesota, Minnesota, USA) and recombinant TGFBI was purified from bacteria as previously described [29 (link)]. Cells were lysed in GST lysis buffer (20 mM Tris-HCl pH 7.4, 50 mM NaCl, 1% NP-40, 10% Glycerol, 0.1% ß-mercaptoethanol, and 10 μg/ml Leupeptin). Lysates were centrifuged and the supernatant was incubated with 10 μg of GST fusion protein and incubated for 2 hours at 4°C with rotation. For in vitro pull-down assays, 2 μg of rTGFBI or 2 μg human plasma fibronectin (Merck Millipore) were incubated with 10 μg of GST fusion protein. Glutathione beads were subsequently washed four times in GST lysis buffer followed by addition of 2X SDS-sample buffer and boiling. Western blot analysis was performed on samples to probe for precipitated proteins.

Due to the difficulty of expressing and purifying intact PuM90, the CDSs of the PuM90 RNA binding domain (PuM90-RBD) and the PuM90 RNA binding domain with three amino acid residues mutated in all eight repeats (PuM90-RBD^AAA) were separately inserted into the pGEX-4T-2 vector (containing the glutathione S-transferase [GST] tag; GE Healthcare Life Science) for in vitro assays. The plasmids (GST empty vector, GST-PuM90-RBD, and GST-PuM90-RBD^AAA) were transformed into E. coli strain BL21 (DE3). A 500-mL culture of E. coli BL21(DE3) cells was grown at 37°C to an optical density at 600 nm of 0.5, after which gene expression was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (Sigma) for 4 h at 28°C. After lysing of cells, the recombinant proteins were purified on glutathione Sepharose beads (Sangon Biotech, Shanghai, China) and eluted with 5 mM glutathione dissolved in Tris-buffered saline. The concentration of purified proteins was determined using a bicinchoninic acid protein assay kit (Sangon Biotech). Protein purity was assessed via SDS-PAGE.

PsAvr3c gene without the regions encoding the signal peptide and RXLR-dEER motif was inserted into the pET32a vector (containing His tag), GmSKRPs and NbSKRP were inserted into the pGEX-4T-2 vector (containing GST tag) (GE Healthcare Life Science). pET32a empty vector, His-PsAvr3c, GST empty vector, GST-GmSKRPs, GST-NbSKRP were expressed in E. coli strain Rosseta2 respectively. The pull-down assay was performed using ProFound pull-down GST protein–protein interaction kit (Pierce) according to the manufacturer’s instructions. The soluble total GST-fusion proteins were incubated with 25 μl glutathione agarose beads (Invitrogen) for 5 h at 4 °C. The beads were washed three times and then incubated with 1 mL bacterial lysates containing His proteins for another 3 h at 4 °C. Then beads were washed three times again, the presence of His proteins was detected by western blot using anti-His antibody.

The primers 5′-CACCCCCGGGCAGCGCTTCTCAGGAGCT-3′ and 5′-GAGACTCGAGTCAGTCTATGTCCTGAAC-3′ (Sigma Genosys Inc., Woodlands, Texas, USA) were used for a polymerase chain reaction (PCR) to amplify a cDNA clone of RANKL. The PCR fragment was subcloned into the pGEX-4T-2 vector (GE Healthcare, Waukesha, WI, USA) after digestion by SmaI and XhoI. The construct was transformed into the BL21 Escherichia coli strain for glutathione-S-transferase (GST) fusion protein expression. The culture was induced with 0.1 mM isopropyl β-d-1-thiogalactopyranoside for 16 h at 20 °C, and the GST-RANKL was purified from bacterial lysate by affinity chromatography on a Glutathione-Sepharose 4B (GE Healthcare) followed by dialysis against multiple changes of PBS. Recombinant GST used as a control was prepared by the same method using an empty pGEX-4T-2 vector. Purified protein was administered to mice by intraperitoneal injections of 250 µg per day for up to 4 days^{14 (link)}. Mice were sacrificed 24 h after the final administration, and samples from these mice were subjected to various assays, as described below.

Human LGALS1/Galectin-1 cDNA (GenBank No. NM_002305) was obtained from DNASU Plasmid Repository (Tempe, AZ) and subcloned into pCMV-Tag 3B vector (Agilent Technologies) with Myc tag and pGEX4T-2 vector (GE Healthcare, Piscataway, NJ) for glutathione S-transferase (GST) fusion. VEGF-Trap_R1R2 (corresponding to aflibercept) cDNA16 (link) was generated as a synthetic gene by IDT (Coralville, IA), and subcloned into the pcDNA6.2/V5-DEST vector (Life Technologies) with V5 tag. The empty pcDNA6.2/V5-DEST vector was used as a Mock transfection control. The GST-fused galectin-1 protein was expressed in an Escherichia coli strain Rosetta-gami 2 (DE3) (Novagen, Madison, WI), and purified through binding to Glutathione Sepharose (GE Healthcare). All constructs were sequence-verified before use.