Chymotrypsin from bovine pancreas

Enzymatic treatment of reticulocyte-enriched samples was performed by incubating 10 µl of the cell suspension with 1 mg/ml trypsin (from bovine pancreas, Sigma), 1 mg/ml chymotrypsin (from bovine pancreas, Sigma) and 0.5 U/ml neuraminidase (from Vibrio cholerae, Sigma) at 37 °C for 1 hour^{69 (link)}. CR1 enzymatic cleavage efficiency was tested by FACSCalibur 4-color flow cytometer (BD Biosciences)^{15 (link)} and neuraminidase treatment was assessed by performing an agglutination test using lectin from the peanut Arachis hypogaea, to detect T-antigen which is exposed after sialic acid cleavage^{69 (link)}.

Reticulocyte-enriched samples were prepared with up to 50% hematocrit. The RBCs were washed with 500 μl of incomplete RPMI 1640 medium twice by centrifugation at 500× g for 3 min at 4 °C. The RBCs were incubated with neuraminidase (from Vibrio cholera, Sigma-Aldrich), trypsin (from bovine pancreas, Sigma-Aldrich) and chymotrypsin (from bovine pancreas, Sigma-Aldrich) at 37 °C on a rotator for 1 h. The trypsin and chymotrypsin-treated RBCs were incubated with a trypsin inhibitor (from the Glycine max soybean, Sigma) at 37 °C for 10 min. The RBC samples were washed twice with 10 ml of incomplete RPMI 1640 medium. The packed cells were prepared at a concentration of 1 × 10⁶cell/ml and used for flow cytometry analysis.

The HRSV M_2–1 protein from strain A was recombinantly expressed in bacteria and purified as described previously (Esperante et al., 2013 ▸ ). The highly purified full-length M_2–1 tetramer was then subjected to limited proteolysis using chymotrypsin from bovine pancreas (Sigma–Aldrich) at a 1:60(w:w) protein:chymotrypsin ratio in 50 mM Tris–HCl pH 8.0 for 90 min at 28°C. The reaction was stopped by adding 1 mM phenylmethylsulfonyl fluoride (PMSF) and subsequently subjected to size-exclusion chromatography on a Superdex 75 column in 20 mM Tris–HCl pH 7.0, 300 mM NaCl. The resulting RBD protein after proteolysis comprises amino acids Ala73–Tyr194. Its molecular weight of 13.6 kDa was confirmed by MALDI–TOF mass spectrometry (Bruker, Daltonics, Billerica, Massachusetts, USA) and the protein elutes as a monomeric peak from size-exclusion chromatography (Supplementary Fig. S2). The protein concentration was determined spectrophotometrically using an extinction coefficient (∊₂₈₀) of 4470 M⁻¹ cm⁻¹.