Production of soluble MERS-CoV S and binding studies

In order to generate soluble MERS-CoV S for binding studies, 293T cells were transfected with an expression vector for the S1 subunit of MERS-CoV S fused to the Fc fragment of human immunoglobulin G (solMERS-S1-Fc). At 24 h post transfection, the culture medium was exchanged and the cells were further incubated for 24 h before culture supernatants were harvested and freed from cellular debris by centrifugation (4,700 x g, 10 min, 4 °C). The clarified supernatants were loaded on Vivaspin protein concentrator columns with a molecular weight cut-off of 30 kDa (Sartorius) and centrifuged at 4,700 x g at 4 °C until the sample was 10-fold concentrated.