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Continuous-Elution Electrophoresis for Purification of the Baculovirus-Expressed Coronavirus Structural Proteins, Rev A

tting using porcine hyperimmune antiserum to TGEV (Stott, 1989) or a TGEV neutralizing monoclonal antibody specific for epitope A of the S-protein (25C9) and non-neutralizing monoclonal antibodies specific for epitope D of the S-protein (44C11) and N-protein (25H7) (Lanza et al., 1995). The protein concentration was estimated with spectrophotometer readings at 280 nm.


Results
By extrapolating the molecular size to prestained protein standards or to native coronavirus proteins from virus (Wesley et al., 1991) cell culture adapted TGEV (Miller strain), the recombinant TGEV M-protein (2831 kDa) was expected to be eluted within fraction range 913 (Figure 1A). Fractions 913 were pooled and concentrated. The presumed M-protein was electrophoresed and detected by Coomassie blue stain (Figure 1B, lane 5) and its virus specificity was confirmed by western blot (Figure 1C, lane 5). After concentration of fractions 913, the final protein concentration was 0.4 mg/ml or 5% of the initial solubilized cell lysate protein.

The recombinant TGEV N-protein (47 kDa) was expected to be within fraction range 2527 (Figure 2A). These fractions were pooled and concentrated. The concentrated sample was analyzed as described for the recombinant TGEV M-protein (Figure 2B, C). The resulting purified N-protein concentration was 0.5 mg/ml, or 8% of the initial baculovirus lysate.

The recombinant TGEV S-protein (150 kDa) was expected to be within fraction range 2030 (Figure 3A). Fractions 2030 were pooled and concentrated. The concentrated S-protein sample was further analyzed as described for the M- and N-proteins (Figure 3B, C). In addition to the major band of 150 kDa,
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