Q sepharose high performance

The DENV virus-like particles (VLPs) were produced and kindly provided by The Native Antigen Company, Kidlington, UK. In short: recombinant dengue virus-like particles consisting of DENV prM and E proteins were transiently expressed in suspension culture adapted HEK293 cells. Three days post transfection culture supernatant was cleared by centrifugation and concentrated by tangential flow filtration. In a first step VLPs were purified by a 2-step discontinuous sucrose gradient with 20% and 40% sucrose densities. After a 6 h spin at 25,000 rpm in a SW28 rotor at 10 °C the 20%–40% interphase was harvested by needle-stick. As a second purification step ion exchange chromatography (Q Sepharose High Performance; GE Healthcare) was used in negative mode once. VLPs were further purified by size exclusion chromatography (Toyopearl HW-65F) which also provided exchange of buffers to storage buffer. Purified DENV1–3 VLPs were stored in 10 mM sodium phosphate, 20 mM sodium citrate, 154 mM sodium chloride, pH 7.4. DENV4 VLPs were stored in Dulbecco’s phosphate buffered saline (DPBS) pH 7.4 containing 30% sucrose. VLP samples were stored at − 80 °C.

The Rba. sphaeroides 2.4.1 strain was grown semi-aerobically as described by Chi et al. (Chi et al. 2014 (link)). After reaching stationary stage, the cultures were harvested and pelleted by centrifugation and then resuspended in buffer A (20 mM tris(hydroxymethyl)-aminomethane (Tris) at pH = 8.0). The photosynthetic membranes were released by ultrasonication and subsequently pelleted by centrifugation. The pelleted material was resuspended in buffer A to optical density OD₈₅₀ ≈ 20 (1 cm path), mixed with lauryl dimethylamine-oxide (LDAO) to a final concentration of ~0.5% (v/v) and gently stirred for 20 min at room temperature (RT). The insoluble material was removed by centrifugation. The complexes were purified by loading the supernatant on an anion exchange column (Q Sepharose High Performance, GE Healthcare) equilibrated with buffer A containing 0.06% LDAO followed by 50 mM gradient steps of NaCl from 150 and 500 mM. The protein-containing fraction was eluted with 300–400 mM NaCl.

Single colonies of V. parahaemolyticus were inoculated in LB medium (Fisher) and grown for 16 h at 30°C.
Exhausted culture broth (1.5 litre) was centrifuged at 6000 r.p.m to eliminate cells. Then it was subjected to fractionation with 60% ammonium sulphate. The precipitated proteins were suspended in Tris-HCl 20 mM pH 7.5, 0,025 mM NaCl, and dialyzed for 24 h at 4°C against repeated changes in the same buffer (after 8 and 16 h). After dialyses, proteins were quantified and used for enzymatic activity and for SDS-PAGE analyses. For fractionation, Ionic Exchange Chromatography (Q sepharose High Performance, GE Healthcare Life Sciences, Uppsala, Sweden) was performed using AKTA Start chromatographyc system (GE Healthcare Life Science, Uppsala, Sweden) as follow: total protein was loaded in 5 ml column. After washing with Tris-HCl 25 mM, 0,025 NaCl pH 7.4, proteins were eluted with NaCl gradient (0,025–0,5 mM). The positive fractions were pooled and observed in SDS PAGE and zimography. Fraction enriched in trypsin like activity was separated using HI Prep 16/60 Sephaclyl S-200 HR (GE Healthcare Life Sciences, Uppsala, Sweden). The sequence of purified protease was determined by automated Edman degradation using a Perkin Elmer protein sequencer.

Xenopus haemoglobin (Hb) was extracted by sonication, as previously described^{27 (link)}. Briefly, blood was centrifuged at 300 × g for 30 min at room temperature (RT). The erythrocyte pellet was washed three times in equal volume of dDPBS and centrifuged at 1,000 × g for 30 min. Then, the erythrocyte pellet was suspended in double the volume of 10 mM Tris-HCl (pH 8.0) and sonicated on ice. Erythrocyte homogenates were incubated at 60 °C for 1 h, and then centrifuged at 2,000 × g for 1 h. An equal volume of 10 mM Tris-HCl (pH 8.0) was added to the supernatant and centrifuged at 2,000 × g for 1 h. The supernatant was dialyzed against 10 mM Tris-HCl (pH 8.0) and then centrifuged at 5,000 × g for 20 min. Anion exchange chromatography (Q Sepharose High Performance; GE Healthcare, Milwaukee, WI, USA) was performed on the Hb solution. Purified Hb was separated using an SDS-PAGE and revealed and stained with Coomassie Brilliant Blue.

To remove humic acids, the DNA was further purified with custom-made Q Sepharose columns as described by Mettel et al., 2010 (link). For each DNA sample, 600 µL of Q Sepharose (Q Sepharose High Performance, GE Healthcare, USA) were aliquoted in 1.5 mL reaction vessels and washed four times in 1 mM potassium phosphate buffer (136 mg/L KH₂PO₄, 228 mg/L K₂HPO₄ × 3 H₂O). After washing, the Q Sepharose pellet was resuspended in 300 µL of the same buffer and transferred into a centrifugal filter unit (Durapore-PVDF 0.45 µm, Merck Millipore Ltd, Germany). The self-made column was packed by centrifugation, and the buffer that passed through the filter membrane was discarded. In the next step, the DNA sample dissolved in NE Buffer was pipetted onto the column and centrifuged for 10 s at 3000 × g, thereby DNA and humic acids were binding to the Q Sepharose. The DNA was eluted by adding 80 µL of a 1.5 M NaCl solution, which was also pushed through the column by centrifugation at 3000 × g for 10 s. The elution step was repeated until the clearly visible brown band of humic acids reached the filter membrane, which usually happened after four repetitions. The DNA in the eluate was desalted by binding it to a silica membrane using the DNA Clean and Concentrator Kit (Zymo Research, Germany) following the manufacturer's protocol.