Vivaspin 20

Cell lysis was done with a high‐pressure cell disrupter (T‐S Series Machine, Constant Systems Limited). Following centrifugation, the supernatant was purified by Ni‐immobilized metal affinity column (IMAC), (Äkta start, GE Healthcare, USA or manual). After loading the supernatant on a HisPrep FF 16/10 or manual packed column (GE Healthcare, USA), the column was washed with 4–5 column volumes (CV) of 20 mm Tris‐HCl followed by 4–5 CV of 2 mm imidazole in 20 mm Tris‐HCl, pH 8. The protein was eluted with 200 mm imidazole in 20 mm Tris‐HCl. After dialysis against 20 mm Tris‐HCl, pH 8, the protein was analyzed by SDS‐PAGE for quality control. Depending on the solubility of the construct, the proteins were concentrated to 200–400 mg mL⁻¹ with centrifugal concentrators (Vivaspin 20, 10 kDa MWCO, GE Healthcare, USA) and then frozen at −20 °C until further use.

Among those annotated xylan hydrolyzing genes, one of the GH11 family genes (Tag No. CM1139) - encoding xylanase was amplified by using the designed primers Xyl-F (5’- GGCCCAAGCTTATGAATCAATTTATTAAT-3’) and Xyl-R (5’- GCGCTCGAGAAGATTGCCGTAAC-3’) (Sites of restriction enzyme HindIII and XhoI are underlined). The PCR products was purified, digested with HindIII and XhoI, and ligated into restricted plasmid pET22b(+). The recombinant vector was transformed into E.coli BL21(DE3) competent cells, and spread onto the LB-agar plate containing 50 μg ml^–1 ampicillin. After incubation at 37 °C for overnight, positive colonies were verified by PCR, and the nucleotides were confirmed by sequencing. Before conducting xylanase expression, 2% of the overnight-incubated cultures were added into 50 ml LB medium with ampicillin and incubate at 37 °C until the OD_600nm reached about 0.5 ~ 0.6. Expression of xylanase was induced with 1.0 mM IPTG, followed by continuous incubation at 22 °C for 16 hrs. The supernatant was collected for activity detection by concentrating through the Vivaspin^® 20 centrifugal concentrator (GE Healthcare), and purified by a Ni-nitrilotriacetic acid (NTA) affinity chromatography column (His SpinTrapTM) (GE Healthcare, UK) based on the fused 6 × His tag.

OMVs were isolated from 250 ml of LB cultures^{32 (link)}. E. coli^pBAD28 and E. coli^pApyr were grown to late-exponential phase (OD₆₀₀ ~0.8–1.0) and removed from culture supernatants by centrifugation. The collected supernatants were filtered (0.22 µm) and concentrated using the Vivaspin 20 concentrators, molecular weight cutoff 50-kDa (GE Healthcare), to eliminate free apyrase (~27 kDa) from the medium. The collected concentrates were then centrifuged at 270,000×g for 3 h at 4 °C to yield crude OMVs preparations that were resuspended in PBS and tested for apyrase activity.

Two hundred μg of purified Sat toxin (1 mL) were added in 4 mL of HEPES solution (10 mM pH 8.3) containing 300 μg FITC (Sigma-Aldrich). The pH of the reaction buffer was adjusted to 8.3 by using sodium bicarbonate. The reaction was carried out on ice in the dark for 3 h under continuous stirring. After this period, the reaction was stopped by the addition of 5 mL of NH₄Cl solution (50 mM). The labeled toxin was concentrated by Vivaspin 20 (50,000 molecular weight cut-off) (GE healthcare) and the free FITC was removed by three consecutive washes with HEPES solution. The internalization of the Sat was visualized by confocal fluorescence microscopy (Leica TCS SP8) and images were obtained via LASX computer software.