Ested pBSKII . The sequence was confirmed by DNA sequencing. The NcoI/BamHI fragment was then subcloned into p416Gal1 (p416Gal1-LUC) for expression in yeast. Cartridge-purified oligonucleotide pairs encoding 14-mer peptides (p370(A), p370(B), p530(A), p530(B), pSGG(A), and pSGG(B)) at a concentration of 5 nM in ten mM Tris-HCl, pH eight, 50 mM NaCl, 1 mM EDTA, pH eight, had been phosphorylated working with polynucleotide kinase, annealed by heating to 95 , and gradually cooling to 25 ( 0.1 /5 s), digested with BamHI/XhoI, and inserted into p416Gal1 LUC digested with the same enzymes. Correct insertion was confirmed by sequencing. For recombinant production of FFL fusion proteins, PacI/XhoI segments from p416Gal1-LUC series constructs were subcloned into pPROEX-LUC. Protein Purification–All Hsp104 variants had been expressed and purified as described elsewhere (19). Ydj1 was purified as described previously (30). For purification of recombinant Ssa1, a Saccharomyces cerevisiae strain (SSA1, ssa2, ssa3, ssa4, and pCAUHSEM-SSA1) was grown at 30 to mid-log phase in YP containing two glucose. The culture was then supplemented with 0.1 volume of ten YP (1 (w/v) yeast extract, 2 (w/v) peptone), two glucose, and 100 M CuSO4, as well as the cells have been allowed to induce overnight. Ssa1 was then purified essentially as described elsewhere (30). For expression and purification of FFL and mutant variants, plasmids were transformed into BL21Codon plus cells, and expression of N-terminal poly-histidine-tagged FFL was induced in mid-log phase with one hundred M 622864-54-4 Technical Information isopropyl 1-thio- -Dgalactopyranoside at 18 overnight. Harvested cells were resuspended in 20 mM Tris, pH eight, 400 mM NaCl, ten mM imidazole, and 1.4 mM -mercaptoethanol and lysed by French press. Poly-histidine-tagged FFL was isolated by chromatography on nickel-nitrilotriacetic acid (Qiagen). Pooled peak fractions had been diluted to two mg/ml, dialyzed twice against 20 mM Tris, pH 8, 50 mM NaCl, 1.four mM -mercaptoethanol, and 10 glycerol, and applied to anion exchange chromatography. Peak fractions had been dialyzedVOLUME 283 Quantity 44 OCTOBER 31,30140 JOURNAL OF BIOLOGICAL CHEMISTRYPeptide and Protein 95130-23-7 manufacturer Binding by Hsptwice against 50 mM Tris, pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.eight M ammonium sulfate, and 2 glycerol, and frozen at 80 . Protein concentrations were determined making use of the Bio-Rad Assay Reagent with bovine serum albumin as a standard. Peptide Synthesis–Peptides arrays had been produced by spot synthesis on cellulose membranes in accordance with the manufacturer’s directions (Intavis, Germany). Soluble peptides had been synthesized at the Sophisticated Protein Technologies Center (Hospital for Sick Kids, Toronto, Canada). Stock peptide solutions had been produced freshly by resuspending to 1 mM in sterile water. Concentrations were determined by measuring absorbance at 280 nm or applying the Bio-Rad Assay Reagent with bovine serum albumin as a normal. Hsp104 Binding to Peptide Arrays–Arrays had been blocked in 1 Blocking Answer (Sigma- Aldrich) diluted in binding buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, ten mM MgCl2, 1 mM dithiothreitol), rinsed 3 occasions in binding buffer, and overlaid with 35 nM Hsp104trap in the presence of 2 mM ATP for 1 h at room temperature. Unbound Hsp104 was removed by extensive washing in binding buffer containing ATP. Bound protein was then transferred to polyvinylidene difluoride utilizing a semidry blotter, and Hsp104 was detected using a rabbit polyclonal antibody. Immunoreactive spots were detected by enhanced.