Strep tactin

E. coli Rosetta 2 containing one of the respective plasmids was cultivated in 500 ml LB medium supplemented with solution M (50x, 1.25 M NaH₂PO₄, 1.25 M KH₂PO₄, 2.5 M NH₄Cl, 0.25 M Na₂SO₄) and solution 5052 (50x, 25% [v/v] glycerol, 10% [w/v] α-lactose monohydrate, 2.5% [w/v] d-glucose) for autoinduction of the expression and antibiotics (34 μg/ml chloramphenicol and 100 μg/ml ampicillin) [44 (link)]. Incubation was done at 26 °C and 120 rpm for 48 h. Subsequent purification of the proteins was performed via Strep-tag II/Strep-Tactin affinity chromatography with 1 ml Strep-Tactin columns (Qiagen, Hilden, Germany) according to Nampally et al. [33 (link)], and the protein concentrations were measured with a Pierce™ BCA Protein Assay Kit (Fisher Scientific, Schwerte, Germany).

Marburg virus GP immunogens used to raise antibodies at Emergent were designed and produced at TSRI. DNA encoding the MARV GPΔmuc ectodomain (residues 1–636 with a mucin deletion of residues 257–425) was cloned into a derivative of the Invitrogen pDisplay vector. In this derivative vector, the PGDFR sequence is replaced by a C-terminal purification tag (either an HA or strep tag). Large-scale production was performed by PEI transfection (Polysciences, Inc MW 25,000) of plasmid into 70% confluent HEK293 GnTI-/- cells (ATCC) in Corning 10-layer Cellstacks. Supernatants were harvested four days post-transfection, concentrated with a Centramate tangential flow system, and affinity purified using Streptactin (Qiagen) or anti-HA 3F10 (Roche) affinity resin. Trimeric GPΔmuc was then isolated by S200 size exclusion chromatography (SEC) in 10 mM Tris, 150 mM NaCl, pH 7.5 (1x TBS). In order to improve furin cleavage processing during expression, which decreased aggregation and improved yield of purified trimers, bulky hydrophobic residues near the furin cleavage site were mutated in the GPΔmuc constructs. Ravn GPΔmuc mutations included F438L, W439A, F445G, F447N and Angola GPΔmuc mutations included W439V, M444A, F445G.