Amicon stirred cell

The designated UF filtered supernatant portion was concentrated 50-fold in 50 mM HEPES buffer in a 400 ml capacity Amicon^® stirred cell (MilliporeSigma, United States) ultradiafiltration system using a 500 kiloDalton (kDa) molecular weight cut off (MWCO) polyethersulfone (PES) ultrafiltration disk (MilliporeSigma, United States). The concentrated retentate was collected and divided into polycarbonate centrifuge tubes (Beckman) before ultracentrifugation at 40,000 RPM for 2 h at 4°C. The pellet was resuspended in 50 mM HEPES buffer and stored at −20°C until further use (Yaron et al., 2000 (link)).

After harvesting, washed larvae (+/- 10 ml) were placed in a 50 ml culture flask with 10 ml culture medium (Earle’s Balanced Salt Solution with 5.3 g/L glucose, 1X Glutamax, 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml gentamicin sulphate and 11.4 U/ml nystatin). Larvae were maintained at 37°C in 5% CO₂. After 48 hrs the medium was discarded and replaced with 10 ml fresh media. At 20 days in vitro (at which point larvae still displayed motility) the culture media was collected, aliquoted, and stored at -80°C until use. For electrophysiology experiments the excretory/secretory extracts were dialysed/buffer exchanged with PBS using an Amicon stirred cell (Merck) with a 3 kDa molecular weight cut-off membrane, in order to remove small molecules that could potentially induce electrophysiological responses that would interfere with the acetylcholine effect (such as glutamate—see [41 (link)].

The strains M. microti-pMV261, M. microti-PstS-1 and M. microti-Ag85 were grown in 250 mL flasks containing 50 mL of Sauton media (composition per liter: 4 g l-asparagine, 0.5 g monopotassium phosphate, 0.5 g magnesium sulphate, 50 mg ferric ammonium citrate, 2 g citric acid, 60 mL glycerol and 0.05% Tween 80), supplemented with 40 mM of glucose and 40 mM of sodium pyruvate at 37 °C with constant stirring at 150 rpm. The kinetic growth was determined every 48 h for 196 h by measuring the O.D. (600 nm) and expressed in absorbance units (A.U.). At the end of the culture, 1 mM of PMSF (phenylmethylsulphonyl fluoride) as a protease inhibitor, 100 µM of EDTA and 0.05% sodium azide were added to the supernatant. The supernatant was concentrated to a volume of 10 mL using an Amicon^® Stirred Cell (Merck™) system and ultrafiltration discs with a nominal molecular weight of 3 kDa (Merck™). It was then filtered again using an Amicon^® Ultra-15 Centrifugal Filter Unit (Merck™). The proteins from the cell extract were recovered by sonicating the centrifuged bacterial pellet with 15 pulses of 100 mV (1 min on/1 min off) at 4 °C in an Ultrasonic Processor (Cole-Parmer^®). To prevent proteolysis, 1 mM of PMSF was added during the sonication. Finally, the concentrated supernatants and cellular extracts of the three strains were recovered and stored at −70 °C until further use.

The supernatant was concentrated by ultrafiltration using an Amicon stirred cell (Merck Millipore) equipped with a 10-kDa membrane to a final volume of 40 ml and extensively dialyzed against 20 mM sodium phosphate and 150 mM NaCl, pH 7.5. The sample was added with NaCl up to 1 M final concentration and the protein was purified using a HiTrap chelating affinity column (5 ml) (GE Healthcare) previously loaded with 100 mM NiCl₂ and equilibrated with 20 mM sodium phosphate, 1 M NaCl, pH 7.5. The column was washed with this buffer until the absorbance value at 280 nm was that of the buffer. rHvRNASET2 was eluted with the same buffer added with 100 mM imidazole; the fractions were equilibrated with 20 mM sodium phosphate and 150 mM NaCl, pH 7.5, by gel-permeation chromatography (PD10 column, GE Healthcare). The amount of protein was determined by using the absorbance intensity at 280 nm and a molar extinction coefficient of 66 mM⁻¹ cm⁻¹ (6 (link)). The recombinant rHvRNASET2 was isolated as a single band at ≈36 kDa with >90% purity as judged by SDS-PAGE analysis: ≈3.5 mg of purified enzyme per liter of fermentation broth was obtained.

Direct Blue 1 (Pontamine Fast Sky Blue 6BX) and Direct Orange 15 (Pontamine Fast Orange 6RN) were obtained from Pylam Products Co. Inc. (Garden City, NY) and used at a working concentration of 10 mg/mL in water. For Direct Orange 15, the low molecular weight components were first removed by ultrafiltration through 100 K membranes with ~200 kPa nitrogen gas (Amicon stirred cell, EMD Millipore Corp., Billerica, MA)—as this fraction demonstrated similar cellulose affinity with Direct Blue 1 [44 (link)]. The residual weight of filter-trapped dye was used to estimate the effective Direct Orange concentration in the filtrate and the stain was then further diluted to the working concentration. The dye mix adsorption isotherm was determined using a series of 1:1 mixtures with increasing concentrations.
Fresh and post-fermentation Populus samples (~10 mg) were suspended in 0.1 mL buffered saline solution (0.3 M Na₃PO₄ and 1.4 mM NaCl at pH 6) and the volume was adjusted to 1.0 ml with deionized water. The samples were incubated with shaking at 70 °C for 6 h, then centrifuged at 10,252×g (10,000 rpm) for 5 min, and the supernatant was collected. Supernatant absorbance was measured using the Lambda 35 UV–Vis Spectrophotometer (PerkinElmer, Waltham, MA), and the calculations for analysis were adapted from Chandra et al. [45 (link)]