Vivaspin column

Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviruses were generated in HEK293FT cells. Cells were seeded in 6-well plates at 1 × 10⁶ cells/mL and directly transfected with pLemir-NS [64 (link)] for RFP delivery, psPAX2 for viral capsid proteins and pCMV-VSV-G [65 (link)] for viral envelope proteins (summarized plasmids are listed in Table 2). After 24 h, medium was collected every 24 h for 120 h in total. After 5 days, medium was centrifuged for 30 min at 2000× g, filtered through 450 nm filters (Sartorius, Göttingen, Germany) and concentrated in Vivaspin columns (Sartorius, Göttingen, Germany).

Spiked wastewater samples (40 mL each) were concentrated by two different methods. First, the PEG (MW 8000)/NaCl precipitation was used as described [20 (link)] with slight modifications (concentration of PEG, resuspension of pellet). Briefly, 10% (w/v) PEG (Carl Roth, Karlsruhe, Germany) and 2.25% NaCl (Carl Roth) were added to the sample and mixed (head over head) at room temperature until complete solving of the additives (15–20 min). Samples were centrifuged at 4 °C and 12,000× g for 2 h in an Avanti HP23 centrifuge (Beckman Coulter, Brea, CA, USA). Supernatant was carefully decanted and sediment was suspended in 500 µL sterile PBS (pH 7.4; Gibco, Paisley, UK). The samples were kept on ice until further processing (within the next 30 min). Second, while the centrifugation of the first half of the sample with PEG-precipitation took place, the other half of the wastewater sample was concentrated with Vivaspin columns (MWCO 10 kDa; Sartorius, Stonehouse, UK) by centrifugation at 4 °C (3300× g) until the concentrate reached a volume of 700–1000 µL. The concentrated supernatant was collected and incubated on ice until RNA preparation of concentrates of both virus enrichment methods as well the spiked material.

Briefly, proteins were overexpressed in E. coli BL21 (DE3) cells by growing at 37°C in LB media containing 50 μg/mL kanamycin to an OD₆₀₀ of 0.4–0.6. Expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. After induction, the cultures were grown at 16°C for an additional 16 hr. The cells were pelleted and lysed via sonication in lysis buffer [50 mM Tris-HCl pH 8.0, 500 mM NaCl, 15 mM Imidazole, 10% Glycerol, 0.1% Triton X- 100, 10 mM β-mercaptoethanol (BME)]. The lysate was cleared by centrifugation, and the supernatant was initially purified using Ni-NTA (Qiagen) beads and eluted with elution buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 200 mM Imidazole, and 10 mM BME). The protein was dialyzed against gel filtration buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mM BME for GST-yCTD and 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 10 mM BME). Finally, proteins were concentrated and ran on a Superdex 200 gel filtration column (GE). Erk2 was purified using a previously published protocol (Khokhlatchev et al., 1997 (link)). Homogeneity of the eluted fractions was determined via Coomassie Brilliant Blue stained SDS-PAGE. Samples were concentrated in vivaspin columns (Sartorius).

Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviruses were generated in HEK293FT cells. Cells were seeded in 6-well plates at 1 × 10⁶ cells/mL and directly transfected with pLenti CMV GFP Neo [22 (link)] for GFP delivery, psPAX2 for viral capsid proteins, and pCMV-VSV-G [22 (link)] for viral envelope proteins (summarized plasmids are listed in Table 1). Also, 16 h after transfection, sodium butyrate containing medium (0.01 M) was added. After 8 h, medium without sodium butyrate was used and collected every 24 h. After 5 days, medium was centrifuged for 30 min at 2000× g, filtered through 450 nm filters (Sartorius, Germany), and concentrated via Vivaspin columns (Sartorius, Göttingen, Germany).

E. coli M15[pREP4] cells (Kan^R) were transformed with roGFP2-Orp1 in pQE60 (carbenicillin resistance, Cn^R) and E. coli KRX cells were transformed with either HyPer-3 or SypHer in pET28a+ (kanamycin resistance, Kan^R). A pre-culture in LB medium (containing 100 μg/ml Cn and/or 50 μg/ml Kan) was inoculated with a colony and grown for 6–7 h at 37°C with vigorous shaking. After incubation, the pre-culture was poured into 50 ml LB medium with antibiotics and grown overnight at 37°C with constant shaking. 500 ml of LB medium containing antibiotics was inoculated with 10–15 ml of the overnight culture and grown at 37°C up to an optical density at 600 nm (OD₆₀₀) of 0.1. The culture was induced at an OD₆₀₀ of 0.6 with either 1 mM IPTG (for expression of roGFP2-Orp1) or 0.1% rhamnose (for expression of HyPer-3 and SypHer) and incubated overnight at RT. The cells were harvested via centrifugation (8,000 rpm, 15 min, 4°C), resuspended (1 g pellet/4 ml buffer) in US buffer (50 mM sodium phosphate buffer, 300 mM NaCl, pH 8.0), and mixed with protease inhibitors (150 nM pepstatin, 40 nM cystatin, 100 μM PMSF) before storage at -20°C. The roGFP2-Orp1, HyPer-3, and SypHer proteins were purified via hexahistidyl affinity chromatography on Ni-NTA material, concentrated using 30 kDa Vivaspin columns (Sartorius, Goettingen), and stored at -20°C with 10% glycerol.

Protease activity was measured by Pierce Protease Assay Kit according to the manufacturer’s instructions with slight modifications. The culture supernatants containing complex BM medium were washed and buffer exchanged using 2 mL Vivaspin columns (Sartorius) as instructed in the manual. Since we were interested in vacuolar proteases active at cultivation pH, the supernatants were reconstituted in 100 mM potassium phosphate buffer, pH = 6. The digestion steps as well as the TNBSA (trinitrobenzenesulfonic acid) development step were incubated for 1 h and overnight, respectively. The buffer exchange was not necessary for samples incubated in M2 medium. However, the incubation times stayed the same.