Mono q 5 50 gl

Purification and pyridylamination of oligosaccharides were performed as described [19 (link), 42 (link), 43 (link)]. Pyridylated (PA)-N-oligosaccharides were separated into neutral N-oligosaccharides and mono−/di−/tri−/tetra-sialylated N-oligosaccharides through an anion-exchange column (Mono Q5/50GL, GE Healthcare) using HPLC and DE52-packed column (Whatman, GE Healthcare) [44 (link)]. Sialylated PA-N-glycans were treated with neuraminidase at 37 °C for 14 h in 50 mM ammonium acetate (pH 5.0) to cleave sialic acids, followed by heating at 100 °C for 5 min and filtering through 0.2 μm spin filter (Ultrafree-MC LG, Millipore). Neutral PA-N-oligosaccharides were analyzed using HPLC as described [19 (link), 42 (link), 43 (link)]. N-oligosaccharide structures were determined by calculating the mannose unit value from NP-HPLC (Takara Bio) and the glucose unit value from RP-HPLC (Takara Bio), as described [20 (link), 45 (link)], also by comparison with known standards and sequential exoglycosidase digestion (see below). PA-N-glycans were quantified as described [44 (link)], and HPLC chromatogram data were analyzed using Unipoint (Gilson), LC station (Shimadzu), and Empower2 (Waters). The workflow of N-oligosaccharides analysis and separation by HPLC is shown in Additional file 2: Figure S1B.

The cDNA fragments encoding the P proteins of two clinical GI.3 huNoVs, the Desert Shield virus DSV395 (GenBank code: U04469.1, VP1 amino acids 227–544) and the VA98115/1998 (GenBank code: AY038598.1, VP1 amino acid 227–538), were chemically synthesized and cloned into expression vector pGEX-6P-1 at EcoRI and SalI/XhoI restriction sites. The P proteins were expressed in E. coli BL21(DE3) as described elsewhere (47 (link)– (link)49 (link)). The resulting GST-P fusion proteins were purified using glutathione-Sepharose 4B (GE Healthcare Life Sciences) according to the manufacturer’s instructions, and the GST tag was removed with Prescission protease at 4°C overnight. The P proteins were further purified by anion ion exchange using Mono Q 5/50 GL (GE Healthcare Life Sciences) and gel-filtration chromatography using Superdex75 (GE Healthcare Life Sciences), in a buffer containing 20 mM HEPES pH 7.5 and 150 mM NaCl (Fig. S1A and B). Purified P proteins were concentrated to 8 mg/mL for crystallization. The P protein mutants of VA115 and DSV were constructed and purified using the same methods as for native P proteins.

The identity of full-length recombinant lymphostatin was confirmed by in-gel protein digest and peptide analysis. Excised gel-bands were incubated at a porcine trypsin:lymphostatin ratio of ∼1:30, in 50 mm ammonium bicarbonate overnight at 32 °C (Promega). Peptides were identified by matrix-assisted laser desorption ionization (MALDI) mass spectroscopy on a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems) using an α-cyano-4-hydroxycinnamic acid matrix. The spectral data were processed using Data Explorer software (Applied Biosystems) and the MASCOT NCBInr database searched against the peptide mass map (Matrix Science). To investigate the domain structure of lymphostatin, purified protein was incubated with trypsin at a ratio of 375:1, at 21 °C, to give limited digestion. Aliquots were removed at 1, 2, 3, and 4 h and the reaction stopped by boiling samples adjusted with 2 mm EDTA and 2 mm PMSF in SDS-PAGE loading buffer. Digest products were separated by SDS-PAGE and individual bands were subjected to in-gel tryptic digestion and MALDI-TOF mass spectroscopy as described above. Peptide masses were compared with the sequence of full-length rLifA using GPMAW 9.2 software, mass tolerance 50 ppm (19 (link)). Fragment F1 was purified to homogeneity from other digest products by ion-exchange chromatography (Mono-Q 5/50 GL; GE Healthcare) as described above.

Isopropyl-1-thio-β-D-galactopyranoside (IPTG) and protease inhibitor cocktail were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anion-exchange chromatography (Mono Q, 5/50 GL) and size-exclusion (Superdex 200, 26/600) columns were purchased from GE healthcare (Fairfield, Connecticut, USA). Teflon beads with a diameter of 2.38 mm were purchased from SmallParts. FSB [(E,E)-1-fluoro-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzene] and curcumin were purchased from Santa Cruz Biotechnology and Sigma-Aldrich, respectively. HS-68 was synthesized according to published procedures^{19 (link)}.

Cysteine residues were introduced by site-directed mutagenesis: E3C in domain 1 (always I27) and N83C in domain 2 if I27, and K83C for I28 (with the numbering relative to a single domain). DNA sequencing confirmed the mutagenesis. I27–I27 and I27–I28 tandems, with the engineered surface cysteines, were expressed as described previously31 (link)47 (link). Labelling was carried out using Alexa Fluor 488 (donor) and Alexa Fluor 594 (acceptor) maleimide (Invitrogen) according to the manufacturer's procedures. Both dyes were mixed simultaneously with reduced protein in equimolar ratios and incubated at 4 °C for ∼10 h. Unreacted dye was removed by gel filtration and the differently labelled variants were separated by ion-exchange chromatography (MonoQ 5/50 GL; GE Healthcare Biosciences AB, Uppsala, Sweden). I27 has two intrinsic cysteines that were not removed as they are buried in the native state and all labelling was carried out on folded protein in native conditions.